Monitoring fetal electrocortical activity during labour for predicting worsening acidemia: a prospective study in the ovine fetus near term.

Abstract

Severe fetal acidemia during labour with arterial pH below 7.00 is associated with increased risk of hypoxic-ischemic brain injury. Electronic fetal heart rate (FHR) monitoring, the mainstay of intrapartum surveillance, has poor specificity for detecting fetal acidemia. We studied brain electrical activity measured with electrocorticogram (ECOG) in the near term ovine fetus subjected to repetitive umbilical cord occlusions (UCO) inducing FHR decelerations, as might be seen in human labour, to delineate the time-course for ECOG changes with worsening acidemia and thereby assess the potential clinical utility of fetal ECOG.
Ten chronically catheterized fetal sheep were studied through a series of mild, moderate and severe UCO until the arterial pH was below 7.00. At a pH of 7.24 ± 0.04, 52 ± 13 min prior to the pH dropping <7.00, spectral edge frequency (SEF) increased to 23 ± 2 Hz from 3 ± 1 Hz during each FHR deceleration (p<0.001) and was correlated to decreases in FHR and in fetal arterial blood pressure during each FHR deceleration (p<0.001).
The UCO-related changes in ECOG occurred in advance of the pH decreasing below 7.00. These ECOG changes may be a protective mechanism suppressing non-essential energy needs when oxygen supply to the fetal brain is decreased acutely. By detecting such "adaptive brain shutdown," the need for delivery in high risk pregnant patients may be more accurately predicted than with FHR monitoring alone. Therefore, monitoring fetal electroencephalogram (EEG, the human equivalent of ECOG) during human labour may be a useful adjunct to FHR monitoring.

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Monitoring Fetal Electrocortical Activity during Labour
for Predicting Worsening Acidemia: A Prospective Study
in the Ovine Fetus Near Term
Martin G. Frasch1,5*, Ashley E. Keen1,2, Robert Gagnon3, Michael G. Ross4, Bryan S. Richardson1,2
1Department of Obstetrics and Gynecology, Lawson Health Research Institute, The University of Western Ontario, London, Ontario, Canada, 2Department of Physiology
and Pharmacology, Lawson Health Research Institute, The University of Western Ontario, London, Ontario, Canada, 3Department of Obstetrics & Gynecology, McGill
University, Quebec, Canada, 4Department of Obstetrics and Gynecology, Geffen School of Medicine at the University of California, Los Angeles, Los Angeles, California,
United States of America, 5Department of Obstetrics and Gynecology, CHU Sainte-Justine Research Centre, Universite ´ de Montre ´al, Quebec, Canada
Abstract
Background: Severe fetal acidemia during labour with arterial pH below 7.00 is associated with increased risk of hypoxic-
ischemic brain injury. Electronic fetal heart rate (FHR) monitoring, the mainstay of intrapartum surveillance, has poor
specificity for detecting fetal acidemia. We studied brain electrical activity measured with electrocorticogram (ECOG) in the
near term ovine fetus subjected to repetitive umbilical cord occlusions (UCO) inducing FHR decelerations, as might be seen
in human labour, to delineate the time-course for ECOG changes with worsening acidemia and thereby assess the potential
clinical utility of fetal ECOG.
Methodology/Principal Findings: Ten chronically catheterized fetal sheep were studied through a series of mild, moderate
and severe UCO until the arterial pH was below 7.00. At a pH of 7.2460.04, 52613 min prior to the pH dropping ,7.00,
spectral edge frequency (SEF) increased to 2362 Hz from 361 Hz during each FHR deceleration (p,0.001) and was
correlated to decreases in FHR and in fetal arterial blood pressure during each FHR deceleration (p,0.001).
Conclusions/Significance: The UCO-related changes in ECOG occurred in advance of the pH decreasing below 7.00. These
ECOG changes may be a protective mechanism suppressing non-essential energy needs when oxygen supply to the fetal
brain is decreased acutely. By detecting such ‘‘adaptive brain shutdown,’’ the need for delivery in high risk pregnant
patients may be more accurately predicted than with FHR monitoring alone. Therefore, monitoring fetal electroenceph-
alogram (EEG, the human equivalent of ECOG) during human labour may be a useful adjunct to FHR monitoring.
Citation: Frasch MG, Keen AE, Gagnon R, Ross MG, Richardson BS (2011) Monitoring Fetal Electrocortical Activity during Labour for Predicting Worsening
Acidemia: A Prospective Study in the Ovine Fetus Near Term. PLoS ONE 6(7): e22100. doi:10.1371/journal.pone.0022100
Editor: Jose M. Belizan, Institute of Clinical Effectiveness and Health Policy, Argentina
Received January 4, 2011; Accepted June 16, 2011; Published July 15, 2011
Copyright: ? 2011 Frasch et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The study was supported by grants to BSR, RG and MR from the Child Health Research Institute, John Patrick Foundation and Department of Obstetrics
and Gynaecology Academic Enrichment Fund, London, and to MGF from CIHR funded Strategic Training Initiative in Research in Reproductive Health Sciences
(STIRRHS), Canada (http://www.stirrhs.ca/fr/user/frasch). The funders had no role in study design, data collection and analysis, decision to publish, or preparation
of the manuscript.
Competing Interests: The authors have read the journal’s policy and have the following conflicts: Financial: Dr. Martin Frasch and Dr. Bryan Richardson are
inventors of related patent applications entitled ‘‘EEG Monitor of Fetal Health’’ including U.S. Patent Application Serial No. 12/532,874 and CA 2681926 National
Stage Entries of PCT/CA08/00580 filed March 28, 2008, with priority to US provisional patent application 60/908,587, filed March 28, 2007. The inventors have
assigned their interests in the patent applications to the Lawson Health Research Institute who is the sole owner responsible for commercialization. The inventors
are entitled to 50% of any net future revenue from commercialization after patenting and commercialization costs are deducted. Lawson Health Research Institute
in all cases will retain rights to the academic and non-commercial use of the technology described in the related patent applications, which therefore does not
alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials as detailed in the guide for authors.
* E-mail: mg.frasch@umontreal.ca
Introduction
Studies of electrocortical activity in the ovine fetus near term
using electrocorticogram (ECOG) recordings reveal alternating
epochs of low-voltage/high frequency (LV/HF) electrocortical
activity with high-voltage/low frequency (HV/LF) ECOG
indicative of behavioural states that relate to cortical neuronal
activity [1–3] (reviewed in [4]). This ECOG state activity
becomes altered in response to induced hypoxia both chronically
by reducing maternal inspired oxygen over several hours [5] and
acutely by occluding the umbilical circulation over several
minutes [6], with disruption in behavioural state cyclicity and
flattening of the ECOG voltage amplitude. These ECOG
changes are paralleled by changes in cerebral metabolism. An
overall decrease in oxygen uptake and increasing reliance on
anaerobic metabolism are likely triggered by critical decreases in
the brain’s oxygenation [6,7].
In studies of cerebral hypoxic-ischemia in adults, there is a
reproducible sequence of changes in cerebral metabolism [8].
Below an upper ischemic flow threshold suppression of synaptic
transmission occurs, but neuronal energy levels and cellular
integrity are maintained. When a lower ischemic flow threshold is
reached, membrane failure occurs that indicates energy depletion
and is closely associated with the development of structural cell
damage. Hence, the initial suppression of synaptic transmission
with a flattening of ECOG as a response to worsening hypoxia
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may be protective by decreasing the energy consumed by Na+-K+-
ATPase involved in the generation of synaptic potentials [8].
Importantly, these ECOG and related metabolic changes within
the brain should occur in advance of asphyxia-mediated brain
injury. Thus, changes in ECOG might prove useful for signaling a
risk of impending brain injury.
Electronic fetal heart rate (FHR) monitoring is the mainstay for
assessing fetal health during labour, because the absence of FHR
decelerations is highly predictive of normal fetal blood gas and pH
at birth [9–12]. Variable-type FHR decelerations due to umbilical
cord compression with acute reduction in fetal oxygenation are the
most common decelerations observed during labour in humans
[13]. When frequent and/or severe, these FHR patterns are
associated with an increased incidence of concerning acidemia in
the newborn [14–16]. However, FHR monitoring is less predictive
of the degree of hypoxia-induced acidemia, i.e., FHR monitoring
has a low positive predictive value for clinically important
metabolic acidosis at birth. Accordingly, the study of the ancillary
monitoring techniques was recommended [12]. Over the past
decade several complimentary monitoring techniques were
assessed including computerized FHR data acquisition and
interpretation, fetal pulse oximetry and ECG waveform analysis
[9–11]. To date, none of these techniques have become ‘standard
of care’ in North America. Hence, there is a clinical need to
improve the existing technologies used for monitoring fetal health
during labour.
We hypothesized that consistent changes in fetal ECOG will
occur well in advance of attaining a severe degree of hypoxic-
acidemia due to suppression of neuronal synaptic activity below
the upper ischemic flow threshold. We studied ECOG in the near
term ovine fetus, a well established model of human pregnancy,
subjected to repetitive umbilical cord occlusions (UCO) leading to
FHR decelerations with worsening acidemia as might be seen in
human labour. Animals were studied through a series of mild,
moderate and severe UCO until fetal arterial pH fell below 7.00,
this being the threshold for severe acidemia in the human newborn
and fetal sheep below which there is increasing risk of hypoxic-
ischemic brain injury [17–22]. Changes in ECOG amplitude and
frequency components were analyzed in relation to the degree of
fetal acidemia as well as the associated cardiovascular responses.
Confirming our hypothesis we found changes in ECOG occurring
in advance of the threshold for severe acidemia with pH,7.00.
This finding has clinical importance, since the feasibility of
recording the electroencephalogram (EEG, the human equivalent
of ECOG) from a scalp electrode during human labour has been
demonstrated [23,24].
Results
Arterial blood gases and pH
epetitive UCO insults resulted in fetal arterial blood gas,
oxygen saturation (O2Sat) and pH changes with each cord
occlusion as well as cumulative changes over the course of the
study as reported (Table 1, [25,26]). The key finding relevant to
interpreting the cardiovascular and ECOG responses herein
presented is that fetal arterial pH values showed a progressive
decrease throughout the UCO series from baseline values of
7.3660.01 to 6.9060.04 following completion of the severe
UCO series (p,0.05). Two animals reached the target pH,7.00
during the moderate UCO series, while the remaining eight
animals took between 20 and 100 minutes during the severe
UCO series to reach the target pH. The animal with the lowest
arterial pH (6.64) died shortly after stopping the repetitive cord
occlusion insults.
Cardiovascular responses
During the baseline control period, FHR averaged 16365 bpm.
While variably increased during the 10 minute periods without
UCO after the mild and moderate UCO series and through the first
hour of recovery after the severe UCO series, none of these changes
were significant (Table 2). During each of the UCO series the FHR
decelerations were of similar degree reaching an average nadir of
8062 bpm. Consequently, the UCO-related depth of FHR
decelerations averaged 8362 bpm (Table 2).
Likewise, during the baseline control period, the ABP averaged
4564 mmHg. The maximum ABP during UCO was of a similar
degree on average for each of the UCO series at 6461 mmHg,
resulting in a UCO-related maximal ABP increase of 1961 mmHg
(Table 2). For the mild UCO series, the ABP we measured at the
time of the nadir of the FHR deceleration (ABPFHR nadir) was higher
on average than the respective maximum ABP during UCO
(ABPmax) at 6768 vs. 6463 mmHg (Table 2).For the moderate and
severe UCO series, ABPFHR nadirwas now lower on average than
the respective ABPmax, at 5464 vs. 6364 mmHg and at 60610 vs.
6566 mmHg (Table 2). That is, while the maximum increase in
ABP during UCO was similar for the three UCO series, the
increased ABP was not sustained and fell somewhat when again
measured at FHRnadirfor the moderate and severe UCO series.
Accordingly, we found no correlation of ABPmaxor maximal ABP
increase (DABPmax) to fetal pH as assessed for the 10 minute
intervals prior to blood sampling during the UCO series for each
animal. However, ABPFHR nadir was found to decrease and
DABPFHR nadirwas found to increase with lower pH, R=0.45
(p=0.01) and R=20.52(p,0.01), respectively, againindicating an
inability to sustain the UCO-related hypertension with worsening
acidemia.
Cerebral electrical responses (ECOG)
During the baseline control period, mean ECOG amplitude
averaged 88613 mV, and did not change significantly as measured
through each of the UCO series (Table 3). Conversely, mean
ECOG 95% SEF which averaged 14.460.4 Hz during the
baseline control period, was significantly decreased as measured
through the moderate and severe UCO series at 11.460.6 Hz and
Table 1. Fetal arterial blood gas, O2Sat and pH
measurements.
pO2
mmHg
pCO2
mmHgO2Sat %pH
Baseline18.26.8 52.76.950.063.1 7.366.01
Mild UCO series
1stUCO9.761.4* 57.361.7*18.364.6*7.326.02
5 min post18.56.952.861.047.263.5 7.326.03
Moderate UCO series
1stUCO10.861.0*57.061.5*19.763.7*7.286.03*
5 min post19.06.8 56.261.3 43.563.97.196.04*
Severe UCO series
1st UCO10.861.9* 58.161.4* 16.464.1*7.216.03*
5 min post 19.461.877.369.3*27.263.2* 6.906.04*
Recovery
1sthr17.46.748.961.0 34.862.2* 7.186.02*
Values are means 6 SEM. N=10. UCO=umbilical cord occlusion.
*, p,0.05 vs. baseline.
doi:10.1371/journal.pone.0022100.t001
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9.660.4 Hz, respectively (both p,0.05) (Table 3). ECOG
amplitude and 95% SEF as measured during the UCO-induced
FHR decelerations were little changed with the mild UCO series,
but were significantly decreased with the severe UCO series at
60612 mV and 9.961.1 Hz, respectively, when compared to the
respective baseline values (both p,0.05) (Table 3). Likewise,
during the severe UCO-induced FHR decelerations, ECOG
amplitude was markedly decreased from that for the 30 seconds
prior to these UCO by 54613 mV, although there was no related
change in the DECOG SEF (Table 3).
For individual animals, mean ECOG amplitude during the
UCO-induced FHR decelerations also decreased with worsening
acidemia and lower pH as assessed at 10 minute intervals prior to
each blood sampling during the UCO series, R=0.51 (p,0.01).
There was no significant relationship between mean ECOG SEF
and fetal pH change when similarly analyzed. Additionally,
DECOG AMP and DECOG SEF, i.e., the relative change in
ECOG amplitude and 95% SEF prior versus during the UCO-
induced FHR decelerations, were both seen to increase with
worsening acidemia and lower pH, R=20.65 and 20.68,
respectively (both p,0.001).
In the course of worsening UCO and associated acidemia we
observed a specific pattern of change in ECOG SEF in relation to
FHR decelerations for all of the animals studied (Figures 1 and 2).
This pattern was visually recognized and defined by the onset of
abrupt increases in ECOG 95% SEF values up to 2362 Hz
towards the end of each UCO-induced FHR deceleration. This
pattern usually lasted no more than 15 to 20 seconds before
rapidly declining to 361 Hz between FHR decelerations. This
SEF ‘spiking’ pattern was detected 52613 minutes (range 1 h
58 min to 25 min) prior to reaching the target pH,7.00. Once
initiated, this pattern continued in all animals until the cord
occlusion insults were stopped. However, the onset of this pattern
in relation to worsening acidosis was variable occurring at a pH of
7.2460.04 on average, but ranging from 7.36 to 7.06 as
determined from the most proximate blood sample (outlined in
Methods). Of note, the onset of this pattern was closely related to
the inability to sustain the UCO-related increase in ABP; ABP was
now either unchanged or decreased as measured at the nadir of
each FHR deceleration when compared to pre-UCO values.
Accordingly, for individual animals the lengths of time over which
the ECOG SEF ‘spiking’ pattern and the hypotensive ABP
responses were seen prior to reaching the target pH,7.00 were
similar and highly correlated, R=0.89 (p,0.001). Most animals
showed the UCO related spikes in ECOG SEF slightly before
showing the ABPFHR nadirhypotensive response.
Discussion
The main finding of this study is the detection of an ECOG
pattern occurring in advance of worsening fetal acidemia and
associated with FHR decelerations induced by repetitive umbilical
cord occlusions as they may occur during human labour.
Monitoring human fetal EEG during labour may potentially be
an ancillary tool to improve the ability to detect worsening fetal
acidemia in advance of brain injury. We propose that the
pathophysiological mechanism producing the observed ECOG
pattern is an acute adaptive response of the fetal brain to suppress
non-essential energy needs during asphyxia. We discuss the
Table 2. Fetal cardiovascular measurements.
Mean FHR, bpm Mean ABP, mmHgFHRnadir, bpm ABPmax, mmHgABP at FHRnadir, bpm
Baseline163654564
Mild UCO series8365*6463*6768*
Post Mild UCO series 1856144864
Mod UCO series 7865* 6364* 5464*
Post Mod UCO series 1716115967
Severe UCO series7864*6566* 60610*
Recovery 1sthr18967 57611
Values are means 6 SEM. N=10. UCO=umbilical cord occlusion; FHR=fetal heart rate (bpm, beats per minute); ABP=arterial blood pressure, mmHg; FHRnadir=UCO-
related nadir of FHR deceleration; ABPmax,=maximum ABP during UCO; ABPFHR nadir=ABP at FHRnadir;
*, p,0.05 vs. baseline.
doi:10.1371/journal.pone.0022100.t002
Table 3. ECOG measurements.
Mean AMP, mV Mean 95% SEF, HzUCO AMP, mVUCO 95% SEF, HzUCO DAMP, mV UCO DSEF, Hz
Baseline 8861314.460.4
Mild UCO series9761413.060.79062714.162.1967
20.860.9
Moderate UCO series9862411.460.6* 81621 11.260.9 1568
54613+
20.561.2
Severe UCO series 98619 9.660.4* 60612* 9.961.1*
21.462.0
Recovery 1sthr9161013.860.4
Values are means 6 SEM. N=8. UCO=umbilical cord occlusion; AMP=amplitude; 95% SEF=95% spectral edge frequency; UCO AMP and 95% SEF=respective ECOG
measurements during UCO; DUCO AMP and SEF=respective ECOG measurement change from 30 seconds prior to each UCO to that during UCO,+p,0.05 for ECOG
amplitude differences during the severe UCO series 30 s prior to the UCO to the amplitude during the UCO;
*p,0.05 vs. baseline.
doi:10.1371/journal.pone.0022100.t003
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correlated changes in arterial blood gases, cardiovascular respons-
es and ECOG in this context.
Arterial blood gases and pH
All fetuses attained a severe metabolic acidemia with base excess
values of 216.661 mmol/L as reported [25,26]. However, the
degree of fetal acidemia after each of the UCO series differed
somewhat amongst the animals as did the actual severity of
acidemia attained. Accordingly, we analyzed the mean cardiovas-
cular and ECOG findings for each of the UCO series, as well as
individual animal findings for the 10 minute intervals prior to each
blood sampling during the UCO series to further assess changes
with worsening acidosis.
Cardiovascular responses
Cord occlusions of one minute duration during mild UCO
series resulted in an immediate fall in FHR to a nadir value of
8365 bpm at or shortly after the end of the occlusion and this did
not change significantly in magnitude during successive occlusions
throughout the UCO series. This finding is similar to that of de
Haan et al. [27] with their study of repetitive UCO of 1 minute
duration every 2.5 minutes in fetal sheep, and indicates that the
chemoreflex mechanisms shown to mediate fetal bradycardia with
acute hypoxia are consistently active despite severe acidosis
[28–30].
Fetal ABP increased during each UCO to a maximum value of
6461 mmHg or by 1961 mmHg on average from baseline
values, and this again did not change significantly in magnitude
throughout the UCO series. This finding was also noted by de
Haan et al. [27] and suggests that the chemoreflex mechanisms
leading to peripheral vasoconstriction are also intact despite severe
fetal acidemia and augment the initial hypertension with
mechanical occlusion of the umbilical arteries [28–30]. However,
this UCO-related increase in ABP was not sustained as indicated
by the lower ABPFHR nadirvalues at the end of each UCO or
shortly thereafter during the moderate and severe UCO series,
and with the fall in ABPFHR nadirvalues well correlated with the
degree of fetal acidemia. This biphasic blood pressure pattern was
likewise noted by de Haan et al. [27]. After the initial UCO-
related increase in ABP they observed a gradual progressive fall
with the minimum blood pressure usually occurring shortly after
release of the occluder, and the recovery time to regain normal
Figure 1. Representative segments of fetal heart rate (BPM, beats per minute) and electrocorticogram (ECOG). Umbilical cord
occlusion (UCO) induced changes are shown in 15 minutes segments of FHR, ECOG amplitude (mV) and 95% spectral edge frequency (SEF, Hz).
Baseline and the 1sthour of recovery are shown for comparison. Note an early emergence of the time correlated changes in FHR decelerations and
increases in ECOG SEF with modest fetal acidemia (pH=7.26). The arrows indicate fetal blood sampling and the corresponding pH values at these
time points.
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blood pressure values was prolonged in the face of worsening
acidemia. The onset and progression of this biphasic blood
pressure response may indicate an inability to maintain the
chemoreflex mediated vasoconstriction or an inability to maintain
cardiac output due to impaired myocardial contractility, or both,
as shown in both humans and sheep with fetal acidemia [31,32].
Cerebral electrical responses: mechanisms of changes in
ECOG amplitude and frequency
Mean ECOG amplitude as measured through each of the mild,
moderate and severe UCO series was marginally increased but not
significantly different from that of the baseline control value. This
can be attributed to an overall increase in time spent in HV/LF
state activity as well as indeterminate voltage/frequency (IV/F)
activity, but then offset by a variable decrease in voltage amplitude
during each UCO in the near term ovine fetus with repetitive
short duration UCO insults [6,33,34].
Mean ECOG amplitude during the UCO-related FHR
decelerations was variably decreased while the change from that
30 seconds prior to each UCO, i.e., DECOG AMP, was
conversely increased. However, this was only significant for the
severe UCO series. This is consistent with an adaptive suppression
of synaptic transmission activity to decrease energy needs with
longer UCO insults [5,6,33,35]. This adaptive metabolic shut-
down by the ovine fetal brain appears to be mediated by
endogenous activation of adenosine A1 receptors during critical
decreases in oxygenation as shown by Hunter et al. with cord
occlusion study during adenosine A1 receptor blockade [5]. The
onset of this response is rapid — within 30 to 60 seconds after
complete cord occlusion — whether measured by decreased
ECOG amplitude or cerebral metabolic rate [5,6,33,35]. Howev-
er, this response will also depend on the integrity of other defense
mechanisms including the ability to redistribute and maintain
increased blood flow to the brain. As such, the significant decrease
in ECOG amplitude during severe UCO in the present study may
be explained by the related hypertensive-hypotensive blood
pressure pattern, which would serve to limit the hypoxia-induced
increases in cerebral blood flow [6,7,36]. The decrease in ECOG
amplitude during UCO and the increase in DECOG AMP
showed a modest relationship to falling arterial pH with R values
of 0.51 and 20.65, respectively. This finding is again similar to
that of de Haan et al. in their study of repetitive UCO where fetal
ECOG intensity decreased continuously with worsening acidemia
[34].
Mean ECOG 95% SEF during each of the UCO series
decreased in a stepwise manner from the baseline control value
consistent with increasing time spent in HV/LF and IV/F activity,
but again offset by the abrupt increases in SEF values towards the
end of each UCO-induced FHR deceleration during the moderate
and/or severe UCO with worsening acidemia. Of note, a study by
Thaler et al. [24] using SEF analysis of fetal EEG during human
labour with variable FHR decelerations but without acidemia
showed voltage amplitude to be increased while SEF was
decreased — consistent with a transition to HV/LF state activity,
albeit in a limited number of healthy subjects.
Mean ECOG 95% SEF during the UCO-related FHR
decelerations was similar to that measured through each of the
respective UCO series and thereby also decreased in a stepwise
Figure 2. Representative segments of correlated changes in fetal heart rate decelerations (BPM, beats per minute) and
electrocorticogram (ECOG) spectral edge frequency (SEF, Hz) spiking pattern. ECOG SEF, FHR and fetal arterial blood pressure (ABP,
mmHg) are shown during a 10 minutes segment demonstrating the correlated changes in FHR decelerations and ECOG SEF spiking pattern during
each umbilical cord occlusion (UCO, indicated by the black bars). Note pathological decreases of ABP during the FHR decelerations.
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manner from the baseline control values, but with no significant
change from that measured 30 seconds prior to each UCO, i.e.,
DECOG SEF. This is again consistent with increasing time spent
in HV/LF and IV/F activity with worsening UCO insults.
However, with worsening UCO and fetal acidemia we also noted
the onset of abrupt increases in SEF of short duration towards the
end of each UCO-induced FHR deceleration in all animals. This
SEF ‘spiking’ pattern was well correlated with worsening of the
biphasic hypertensive-hypotensive blood pressure response. The
appearance of the SEF ‘spiking’ pattern is likely triggered by a
further limitation in the hypoxia-induced increases in cerebral
blood flow due to the worsening hypotension or worsening
acidemia. The emergence of this SEF ‘spiking’ pattern suggests
that neuronal activities are being differentially affected and may
reflect the relative dominance of neocortical c-aminobutyric acid
(GABA)ergic inhibitory interneuron activity that is capable of high
frequency oscillations [37]. This might occur due to the active
participation in suppression of synaptic transmission by excitatory
neurons, or due to hierarchal ‘shutdown’ of excitatory neurons in
advance of inhibitory interneurons when cerebral oxygenation
becomes rate-limiting for metabolic activity, since GABAergic
signaling is less energy demanding [38]. The appearance of the
SEF ‘spiking’ pattern with worsening acidemia and the increase in
DECOG SEF with falling pH (as assessed for the 10 minutes prior
to each blood sampling during the UCO series) also support a link
between ECOG alterations and systemic acidemia. However, the
DECOG SEF-pH relationship was modest, with an R value of
20.68, and the onset of the SEF ‘spiking’ pattern in relation to
worsening acidosis was variable ranging from pH 7.36 to 7.06 and
appearing as early as 1 h 58 min to 25 min prior to attaining the
target pH,7.00.
The adaptive cardiovascular and ECOG responses to
worsening acidemia are individually specific, and likely
dependent on previous hypoxic-asphyxic exposures
Despite the similar UCO insults, animals showed differing time
courses for metabolic deterioration and for the UCO-related
changes in ECOG. Similar findings were noted by de Haan et al.
in their study of repetitive UCO [27,34]. They reported a
relationship to cardiovascular deterioration as reflected by onset
and progressive worsening in the biphasic hypertensive-hypoten-
sive blood pressure response. This could involve the vasoconstric-
tor response to acute hypoxia and thereby the ability to
redistribute blood flow to the brain, which has been shown to be
diminished in the ovine fetus with prior hypoxic exposure [39].
With repetitive UCO as studied, a diminished redistribution of
blood flow to the brain might lead to earlier onset of UCO-related
changes in ECOG, while a diminished redistribution of blood flow
away from carcass tissues might slow the buildup of lactic acid and
thereby mitigate the fall in systemic pH. While these individual
differences should not alter the principal observation of the
reported ECOG pattern with repetitive UCO and worsening
acidemia, the time course for emergence of this pattern requires
further study in advance of clinical application.
Cerebral electrical responses (ECOG): translational
implications
Overall, our data provide support for a link between ECOG
alterations and systemic acidemia in the ovine fetus near term
subjected to repetitive cord occlusions. Cerebral adaptive
circulatory and metabolic mechanisms are capable of rapidly
suppressing synaptic transmission to reduce non-essential energy
needs when oxygen supply to the fetal brain is decreased acutely
[5,6,33,35]. In this regard, the animals in the present study showed
a rapid normalization of ECOG during the recovery period except
for the animal with the lowest pH, which died. The fact that we
found no evidence of seizure-like activity strongly suggests that the
observed ECOG changes were adaptive rather than reflecting
evolving cerebral injury.
Although individual differences in emergence of the SEF
‘spiking’ pattern were seen, the main clinical implication of our
findings remains that the UCO-related changes in ECOG SEF
occurred well in advance of the threshold for severe acidemia at
which increased risk for asphyxia-mediated brain injury is
reported [17–21,40]. Given the technologic advances for moni-
toring fetal EEG during human labour, fetal EEG may be a useful
adjunct to electronic FHR monitoring for signaling ‘adaptive
metabolic shutdown’ of the fetal brain, and thereby the need for
delivery in high risk pregnant patients [24].
Materials and Methods
Surgical Preparation
Ten near term (12561 days gestation) fetal sheep of mixed
breed were surgically instrumented (term=145 days, Table 4).
The anesthetic and surgical procedure and postoperative care of
the animals were performed as published [41]. Anesthesia was
induced with an injection of sodium thiopental (1 g in 40 mL
solution; Abott Laboratories Ltd., Montreal, Canada) into the
maternal jugular vein and maintained throughout surgery with 1–
1.5% isoflurane in oxygen (Halocarbon Laboratories, Hackensack,
NJ). Using sterile technique under general anesthesia, a midline
incision was made in the lower abdominal wall, and the uterus was
palpated to determine fetal number and position. The upper body
of the fetus and proximal portion of the umbilical cord were
exteriorized through an incision in the uterine wall. Polyvinyl
catheters were placed in the right and left brachiocephalic arteries
and advanced into the ascending aorta as well as the right
brachiocephalic vein and advanced into the superior vena cava.
Stainless steel electrodes were implanted over the sternum to
record electrocardiographic (ECG) activity. To record ECOG, the
stainless steel ECOG electrodes were implanted biparietally on the
dura through small burr holes in the skull bone ,2 mm in
diameter made with a hand drill. These burr holes were placed
,1–1.5 cm lateral to the junction of the sagittal and lambdoid
sutures with care taken to avoid puncturing the dura. The bared
Table 4. Subject information.
Number of fetusesWeight (kg)Gender
13.1Male
21.9 Male
1 2.7Male
22.3 Female
13.6 Female
1 2.2Female
12.8 Female
1 2.8Male
1 3.3 Female
1 3.3Female
Mean6SEM1.260.1 2.860.2
doi:10.1371/journal.pone.0022100.t004
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portion of the wire to each electrode was rolled into a small ball
and then inserted into each burr hole to rest on the dura with a
small plastic disk then covering each burr hole. This plastic disk
was then thinly coated with tissue adhesive (cyanoacrylate) and
held in position against the skull bone surrounding each burr hole
until firmly adherent. The reference electrode was then placed in
the loose connective tissue in the midline overlying the occipital
bone at the back of the skull such that the bared wire beyond the
knot was embedded. An inflatable silicone occluder cuff was
positioned around the proximal portion of the umbilical cord and
secured to the abdominal skin. Once the fetus was returned to the
uterus, a catheter was placed in the amniotic fluid cavity and
another in the maternal femoral vein. Antibiotics were adminis-
tered intravenously to the mother (0.2 g trimethoprim and 1.2 g
sulfadoxine, Schering Canada Inc., Pointe-Claire, Canada) and
the fetus and into the amniotic cavity (1 million IU penicillin G
sodium, Pharmaceutical Partners of Canada, Richmond Hill,
Canada). Amniotic fluid lost during surgery was replaced with
warm saline. The uterus and abdominal wall incisions were
sutured in layers and the catheters exteriorized through the
maternal flank and secured to the back of the ewe in a plastic
pouch.
Animals were allowed a 3–4 day postoperative period prior to
experimentation. During this period, postoperative daily antibiotic
prophylaxis was continued: 1) to the ewe via maternal femoral
catheter (6 cc Trivetrin, Schering-Plough, Kenilworth, NJ) and 2)
to the fetus via the brachiocephalic vein and amniotic cavity
(1,000,000 IU penicillin G sodium, Pharmaceutical Partners of
Canada, Richmond Hill, Canada). Arterial blood was sampled for
evaluation of maternal and fetal condition and catheters were
flushed with heparinized saline to maintain patency. 10,000 USP
heparin units were dissolved in 250 cc isotonic NaCl. Animal care
followed the guidelines of the Canadian Council on Animal Care
and was approved by the University of Western Ontario Council
on Animal Care.
Experimental protocol
Animals were studied through a 1 to 2 hour baseline period, an
experimental period of repetitive UCO with worsening acidemia,
and were then allowed to recover overnight. The fetal arterial and
amniotic pressures, ECOG and ECG were monitored continu-
ously through the control and experimental periods, and first hour
of the recovery period.
After the baseline period which began at ,0800, repetitive
UCO were performed with increasing frequency until severe
fetal acidemia was detected (arterial pH,7.00), at which time
the UCO were stopped. Complete UCO was induced by
inflation of the occluder cuff with ,5 mL saline solution, the
exact volumes having been determined by visual inspection and
testing at the time of surgery for each animal. During the first
hour a mild UCO series was performed consisting of cord
occlusion lasting for 1 minute and repeating every 5 minutes.
During the second hour a moderate UCO series was performed
consisting of cord occlusion for 1 minute duration and repeating
every 3 minutes. During the third hour a severe UCO series was
performed consisting of cord occlusion for 1 minute duration,
repeated every 2 minutes, and this series was continued until the
targeted fetal arterial pH was attained. Following the mild as
well as the moderate UCO series 10 minute periods with no
UCO were undertaken, during which fetal arterial blood was
sampled and arterial blood pressure, ECOG, and ECG data
were recorded in the absence of fetal heart rate decelerations.
After attaining the targeted fetal arterial pH of ,7.00 and
stopping the repetitive UCO, animals were allowed to recover
for ,24 hours.
Fetal arterial blood samples were obtained during the baseline
period (3 mL), at the end of the 1stUCO of each UCO series
(1 mL), and ,5 minutes after each UCO series (3 mL). In
addition, fetal arterial blood samples were obtained between UCO
at ,20 and 40 minutes of the moderate and severe UCO series
(1 mL), and at 1, 2 and 24 hours of recovery (3 mL). Maternal
venous blood samples were also obtained during the baseline
period, and at 1 and 24 hours of recovery (3 mL). All fetal arterial
blood samples were analyzed for blood gas values, pH, and O2Sat
with an ABL-725 blood gas analyzer (Radiometer Medical,
Copenhagen, Denmark) with temperature corrected to 39.0uC.
The amount of blood withdrawn from each fetus was replaced
with maternal blood at the end of day 1.
After the 24 hour recovery blood sample, the ewe and the fetus
were killed by an overdose of barbiturate (30 mg sodium
pentobarbital IV, MTC Pharmaceuticals, Cambridge, Canada)
and a post mortem was carried out during which fetal gender and
weight were determined, and the location and function of the
umbilical cord cuff was confirmed. The fetal brain was then
perfusion-fixed with 500 mL of cold saline followed by 500 mL of
4% paraformaldehyde and processed for histochemical analysis as
reported [25].
Data acquisition and analysis
Arterial and amniotic pressures were measured continuously
using Statham pressure transducers (P23 ID; Gould Inc., Oxnard,
CA). Arterial blood pressure (ABP) was determined as the
difference between instantaneous values of arterial and amniotic
pressures. A PowerLab system was used for data acquisition and
analysis (Chart 5 For Windows, ADInstruments Pty Ltd, Castle
Hill, Australia). Arterial and amniotic pressures, ECG and ECOG
were recorded and digitized at 1000 Hz. For ECG, a 60 Hz notch
filter was applied, while for ECOG, a band pass 0.3–30 Hz filter
was used. FHR was triggered and calculated online from arterial
pressure systolic peaks.
Average values of FHR and ABP were calculated from
recordings through the baseline period, the 10 minute periods
without UCO after the mild and moderate UCO series, and
through the first hour of recovery after the severe UCO series as
reported [26]. For each UCO we calculated the related nadir of
the FHR deceleration (FHRnadir) and the maximum ABP
(ABPmax) during that UCO, as well as the ABP at the nadir of
the FHR deceleration (ABPFHR nadir). The UCO - related depth of
FHR deceleration (DFHR) and maximal ABP increase (DABPmax)
as well as the ABP change at the nadir of the FHR deceleration
(DABPFHR nadir) were then calculated as the change from that
animal’s respective baseline period values. Averaged values for
these UCO-related FHR and ABP changes were then determined
for each animal for each UCO series as well as for the 10 minute
intervals prior to each blood sampling during the UCO series.
Prior to the ECOG analysis, the ECOG signal was sampled
down to 100 Hz. Subsequently, the voltage amplitude and 95%
spectral edge frequency (SEF, the ECOG frequency below which
95% of ECOG spectral power is found), were calculated over 4 s
intervals (Spektralparameter, GJB Datentechnik GmbH, Lange-
wiesen, Germany). For each animal, average values for ECOG
amplitude and 95% SEF were then determined from recordings
through the baseline period, each of the UCO series and the first
hour of recovery. For each UCO-induced FHR deceleration, we
also determined the related ECOG amplitude and 95% SEF (i.e.,
that occurring from the onset of the deceleration to approximately
60 seconds thereafter). Relative changes in ECOG amplitude and
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95% SEF were also determined (DECOG AMP and DECOG
SEF) as the differences between the mean ECOG amplitude and
95% SEF during the 30 seconds prior to each UCO and the mean
UCO-induced FHR deceleration values. Average values for these
ECOG measurements during cord occlusions were then deter-
mined for each animal for each UCO series as well as for the
10 minute intervals prior to each blood sampling during the UCO
series. The 10 minute interval ECOG values were then correlated
to the measured pH values to better delineate the utility of fetal
ECOG for predicting metabolic deterioration with repetitive
UCO and worsening acidemia.
Statistical analyses
Normal data distribution was tested using Kolmogorov-Smirnov
test. Blood gas, O2Sat and pH measurements in response to
repetitive cord occlusions were compared to baseline values by one
way repeated measures ANOVA with Bonferroni correction for
multiple comparisons. FHR, ABP, and ECOG values in response
to cord occlusions were analyzed by Friedman repeated measures
ANOVA on ranks with Student-Newman-Keuls correction for
multiple comparisons. Pearson or Spearman correlation analysis
were performed as appropriate and R values are presented where
p ,0.05 (SigmaStat, Systat Software, Inc., San Jose, Ca). All
values are expressed as means 6 SEM. Statistical significance was
assumed for p ,0.05. Not all measurements were obtained for
each animal for all time points due to catheter and/or ECOG
electrode difficulties as well as differences in the inter-animal rates
of deterioration and recovery from UCO (see Results).
Acknowledgments
We wish to thank Brad Matushewski, Jeremy McCallum, Andrew Prout,
and Maria Sinacori, all of whom provided technical support for these
studies.
Author Contributions
Conceived and designed the experiments: RG MR BSR. Performed the
experiments: MGF MR BSR. Analyzed the data: MGF AEK BSR.
Contributed reagents/materials/analysis tools: MGF AEK RG MR BSR.
Wrote the paper: MGF BSR.
References
1. Ioffe S, Jansen AH, Russell BJ, Chernick V (1980) Sleep, wakefulness and the
monosynaptic reflex in fetal and newborn lambs. Pflugers Arch 388: 149–
157.
2. Ruckebusch Y, Gaujoux M, Eghbali B (1977) Sleep cycles and kinesis in the
foetal lamb. Electroencephalogr Clin Neurophysiol 42: 226–237.
3. Szeto HH, Hinman DJ (1985) Prenatal development of sleep-wake patterns in
sheep. Sleep 8: 347–355.
4. Richardson B, Gagnon R (2008) Behavioural state activity and fetal health &
development. In: Creasy RK, Resnik R, eds. Maternal-fetal medicine. 6th ed.
Philadelphia: WB Saunders Co. pp 171–179.
5. Hunter CJ, Bennet L, Power GG, Roelfsema V, Blood AB, et al. (2003) Key
neuroprotective role for endogenous adenosine A1 receptor activation during
asphyxia in the fetal sheep. Stroke 34: 2240–2245.
6. Kaneko M, White S, Homan J, Richardson B (2003) Cerebral blood flow and
metabolism in relation to electrocortical activity with severe umbilical cord
occlusion in the near-term ovine fetus. Am J Obstet Gynecol 188: 961–972.
7. Richardson BS, Rurak D, Patrick JE, Homan J, Carmichael L (1989) Cerebral
oxidative metabolism during sustained hypoxaemia in fetal sheep. J Dev Physiol
11: 37–43.
8. Astrup J (1982) Energy-requiring cell functions in the ischemic brain. Their
critical supply and possible inhibition in protective therapy. J Neurosurg 56:
482–497.
9. Liston R, Crane J, Hamilton E, Hughes O, Kuling S, et al. (2002) Fetal health
surveillance in labour. J Obstet Gynaecol Can 24: 250–276.
10. Liston R, Crane J, Hughes O, Kuling S, MacKinnon C, et al. (2002) Fetal health
surveillance in labour. J Obstet Gynaecol Can 24: 342–355.
11. Liston R, Sawchuck D, Young D (2007) Fetal health surveillance: antepartum
and intrapartum consensus guideline. J Obstet Gynaecol Can 29: S3–56.
12. (1997) Electronic fetal heart rate monitoring: research guidelines for interpre-
tation. National Institute of Child Health and Human Development Research
Planning Workshop. Am J Obstet Gynecol 177: 1385–1390.
13. American College of Obstetricians and Gynecologists. Fetal heart rate patterns:
Monitoring, interpretation, and management. Technical Bulletin [207]. 1995.
Washington (DC).
14. Ozden S, Demirci F (1999) Significance for fetal outcome of poor prognostic
features in fetal heart rate traces with variable decelerations. Arch Gynecol
Obstet 262: 141–149.
15. Krebs HB, Petres RE, Dunn LJ (1983) Intrapartum fetal heart rate monitoring.
VIII. Atypical variable decelerations. Am J Obstet Gynecol 145: 297–305.
16. Tortosa MN, Acien P (1990) Evaluation of variable decelerations of fetal heart
rate with the deceleration index: influence of associated abnormal parameters
and their relation to the state and evolution of the newborn. Eur J Obstet
Gynecol Reprod Biol 34: 235–245.
17. Goldaber KG, Gilstrap LC, III, Leveno KJ, Dax JS, McIntire DD (1991)
Pathologic fetal acidemia. Obstet Gynecol 78: 1103–1107.
18. Low JA, Galbraith RS, Muir DW, Killen HL, Pater EA, et al. (1984) Factors
associated with motor and cognitive deficits in children after intrapartum fetal
hypoxia. Am J Obstet Gynecol 148: 533–539.
19. Winkler CL, Hauth JC, Tucker JM, Owen J, Brumfield CG (1991) Neonatal
complications at term as related to the degree of umbilical artery acidemia.
Am J Obstet Gynecol 164: 637–641.
20. American College of Obstetricians and Gynecologists. Use and abuse of the
Apgar Score. Committee Opinion [174]. 1996. Washington (DC), The College.
21. Gunn AJ, Parer JT, Mallard EC, Williams CE, Gluckman PD (1992) Cerebral
histologic and electrocorticographic changes after asphyxia in fetal sheep.
Pediatr Res 31: 486–491.
22. American College of Obstetricians and Gynecologists (2010) Practice bulletin
no. 116: Management of intrapartum fetal heart rate tracings. Obstet Gynecol
116: 1232–1240.
23. Sokol RJ, Rosen MG, Chik L (1977) Fetal electroencephalographic monitoring
related to infant outcome. Am J Obstet Gynecol 127: 329–330.
24. Thaler I, Boldes R, Timor-Tritsch I (2000) Real-time spectral analysis of the
fetal EEG: a new approach to monitoring sleep states and fetal condition during
labor. Pediatr Res 48: 340–345.
25. Prout AP, Frasch MG, Veldhuizen RA, Hammond R, Ross MG, et al. (2010)
Systemic and cerebral inflammatory response to umbilical cord occlusions with
worsening acidosis in the ovine fetus. Am J Obstet Gynecol 202: 82–89.
26. Frasch MG, Mansano RZ, McPhaul L, Gagnon R, Richardson BS, et al. (2009)
Measures of acidosis with repetitive umbilical cord occlusions leading to fetal
asphyxia in the near-term ovine fetus. Am J Obstet Gynecol 200: 200–207.
27. de Haan HH, Gunn AJ, Gluckman PD (1997) Fetal heart rate changes do not
reflect cardiovascular deterioration during brief repeated umbilical cord
occlusions in near-term fetal lambs. Am J Obstet Gynecol 176: 8–17.
28. Itskovitz J, LaGamma EF, Rudolph AM (1983) Heart rate and blood pressure
responses to umbilical cord compression in fetal lambs with special reference to
the mechanism of variable deceleration. Am J Obstet Gynecol 147: 451–457.
29. Giussani DA, Spencer JA, Moore PJ, Bennet L, Hanson MA (1993) Afferent and
efferent components of the cardiovascular reflex responses to acute hypoxia in
term fetal sheep. J Physiol 461: 431–449.
30. Giussani DA, Spencer JAD, Hanson MA (1994) Fetal cardiovascular reflex
responses to hypoxaemia. Fetal Maternal Med Rev 6: 17–37.
31. Lewinsky RM (1994) Cardiac systolic time intervals and other parameters of
myocardial contractility as indices of fetal acid-base status. Baillieres Clin Obstet
Gynaecol 8: 663–681.
32. Fisher DJ (1986) Acidaemia reduces cardiac output and left ventricular
contractility in conscious lambs. J Dev Physiol 8: 23–31.
33. Kawagoe Y, Green L, White S, Richardson B (1999) Intermittent umbilical cord
occlusion in the ovine fetus near term: effects on behavioral state activity.
Am J Obstet Gynecol 181: 1520–1529.
34. de Haan HH, Gunn AJ, Williams CE, Gluckman PD (1997) Brief repeated
umbilical cord occlusions cause sustained cytotoxic cerebral edema and focal
infarcts in near-term fetal lambs. Pediatr Res 41: 96–104.
35. Hunter CJ, Blood AB, Power GG (2003) Cerebral metabolism during cord
occlusion and hypoxia in the fetal sheep: a novel method of continuous
measurement based on heat production. J Physiol 552: 241–251.
36. Lou HC, Lassen NA, Tweed WA, Johnson G, Jones M, et al. (1979) Pressure
passive cerebral blood flow and breakdown of the blood-brain barrier in
experimental fetal asphyxia. Acta Paediatr Scand 68: 57–63.
37. Sejnowski TJ, Paulsen O (2006) Network oscillations: emerging computational
principles. J Neurosci 26: 1673–1676.
38. Waldvogel D, van GP, Muellbacher W, Ziemann U, Immisch I, et al. (2000)
The relative metabolic demand of inhibition and excitation. Nature 406:
995–998.
39. Gardner DS, Fowden AL, Giussani DA (2002) Adverse intrauterine conditions
diminish the fetal defense against acute hypoxia by increasing nitric oxide
activity. Circulation 106: 2278–2283.
Fetal ECOG in Worsening Acidemia
PLoS ONE | www.plosone.org8 July 2011 | Volume 6 | Issue 7 | e22100

Page 9

40. Mallard EC, Williams CE, Johnston BM, Gunning MI, Davis S, et al. (1995)
Repeated episodes of umbilical cord occlusion in fetal sheep lead to preferential
damage to the striatum and sensitize the heart to further insults. Pediatr Res 37:
707–713.
41. Green LR, Homan J, White SE, Richardson BS (1999) Cardiovascular and
metabolic responses to intermittent umbilical cord occlusion in the preterm
ovine fetus. J Soc Gynecol Investig 6: 56–63.
Fetal ECOG in Worsening Acidemia
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