Assessment and quantification of foetal electrocardiography and heart rate variability of normal foetuses from early to late gestational periods 2

Materials and methods 82 2 Methodology 2.1 Foetal ECG acquisition procedures The fECG was recorded using a non-invasive system by attaching three cutaneous electrodes on the maternal

Materials and methods 80

CHAPTER 6 MATERIALS AND METHODS

1 Patient selection

1.1 Study subjects

This is a longitudinal study whereby serial electrocardiograms (ECG) from a

cohort of 100 healthy foetuses were measured from 18 to 41 weeks of gestation The

women who participated in this study had singleton pregnancies and gave informed

consent They were recruited at about 18 weeks of pregnancy from an antenatal clinic

at the National University Hospital Foetal ECG (fECG) recordings were monitored at

the first visit and at each subsequent antenatal visit to the clinic until the final visit

before delivery The interval between antenatal visits ranged from one to four weeks

depending on the stage of the pregnancy

1.2 Exclusion criteria

Women whose foetuses exhibited arrhythmia, IUGR (intrauterine growth

restriction), congenital heart disease, or in whom maternal hypertension, diabetes or

SLE (Systemic Lupus Erythematosus) was present, were excluded from the study

1.3 Patient withdrawal

Initially, 115 pregnant women were recruited progressively After the

exclusion of those with the above-mentioned conditions, 100 women were left to

participate in the study Out of these 100 remaining participants, nine did not

complete the study due to reasons such as delivery in another hospital or country and

foetal loss In addition, 23 women refused monitoring on at least one follow-up visit

due to personal reasons

Materials and methods 82

2 Methodology

2.1 Foetal ECG acquisition procedures

The fECG was recorded using a non-invasive system by attaching three

cutaneous electrodes on the maternal abdominal surface The three electrodes are

namely, one designated reference electrode (black) and two other recording

electrodes (red), one of which is positive and the other negative The three disposable

electrodes (3M Red Dot 2237) for recording fECG were placed in an equilateral

triangle formation on the maternal abdomen – one electrode each at the right and left

hypochondriac area and one electrode just above the symphysis pubis (Figure 6-1)

The reference electrode was placed either at the right or left hypochondriac area while

the recording electrodes are placed on the two remaining sites The location of the

reference electrode was determined based on the quality of the signal All three

electrodes were connected to a single channel digital recorder (patient unit) with a

sampling rate of 300 Hz (Figure 6-2) The patient unit was connected via a fiber optic

data link to the computer that operated the FEMO system software The whole set-up

is illustrated in Figure 6-3

To reduce skin impedance, the three areas of skin contact were gently abraded

to remove dead surface skin cells After electrode placement, a skinprep analyzer

(Union skinprep 3211D) was used to ensure that the electrode impedance was below

the recommended 5 kΩ The women were rested in a semi-recumbent position for at

least 5 minutes prior to the actual fECG recording, which lasted for 10 minutes All

recording sessions took place between 0900 hrs and 1700 hrs

Electrode Navel

Figure 6-1: Electrode placement for abdominal fECG recording

Materials and methods 84

Figure 6-2: Patient unit of FEMO system and 3 recording electrodes

Figure 6-3: Setup of FEMO system

Materials and methods 86

2.2 Foetal ECG equipment description and operation

The abdominal fECG was measured and processed instantaneously by a

specifically-designed computerized system, FEMO (Medco Electronic Systems Ltd.,

Israel) FEMO system is an advanced, non-invasive device that detects the

superimposed foetal and maternal ECG (mECG) signals from the maternal abdomen,

then separates and processes the two signals It operates in real-time (delay not longer

than 0.06 seconds), displaying the true beat-to-beat foetal heart rate and the averaged

fECG complex

In abdominal ECG processing, the single most important problem is the

subtraction of the mECG template from the combined foetal-maternal ECG signals

obtained from the abdominal recording This subtraction is most vulnerable to

computational errors and must be performed at the precise moment Otherwise, the

residual value of the maternal contribution will be larger than the foetal contribution

The core of the FEMO fECG detection algorithm (patented) is based on cancellation

of the maternal contribution in both the first and second derivatives of the abdominal

signal In contrast to other cancellation procedures, which are generally performed in

the abdominal ECG signal itself The processing of the two foetal-maternal

derivatives as two independent parallel data channels reduces computational errors

arising from subtraction of the maternal signals This is because these errors are

unlikely to occur simultaneously in both channels Thus the double computation and

the combination of the resulting functions reduce the number of detection errors,

thereby successfully producing accurate fECG signals without any maternal

contribution In addition, the two derivatives also eliminate the influence of baseline

drift, and the derivation procedure provides additional filtering to differentiate the

maternal and foetal ECG

A low pass filter with a cut-off frequency of 25 Hz provided an output

containing pure maternal signals, while a 110 Hz low pass filter transferred both

maternal and foetal signals The first derivative was then computed by recursive

integration The second derivative was calculated directly by an approximation based

on the undetermined coefficients (Lagrange) interpolation method The exact

locations of maternal R waves were determined by a local fine adjustment procedure,

and the mECG template M was constructed After separately subtracting M from the

1st and 2nd derivatives, the results were summed into a combined signal from which

the foetal complexes were detected after undergoing a smoothing procedure A

simplified flow chart of the algorithm is shown in Figure 6-4

The reliability and accuracy of the FEMO system has been tested against the

‘gold standard’ scalp electrode fECG measured during labour Excellent agreement is

obtained both in foetal heart rate (Figure 6-5a) and in the foetal ECG morphology

(Figure 6-5b), with a correlation of 0.9 and significance of p<0.001 (Karin J et al,

1994)

Materials and methods 88

Subtraction of maternal contribution, M

Raw abdominal ECG data

Calculate average foetal ECG complex

Calculate foetal RR interval series Summation of 1st and 2nd derivatives

Figure 6-4: Block diagram of R wave detection algorithm in FEMO software

A/D- Analogue to digital converter; M- maternal ECG signal; MF’ and MF’’- 1st and

2nd derivatives containing both maternal and foetal ECG signals; F’ and F’’- Foetal

ECG signals after subtraction of M from MF’ and MF’’

Figure 6-5a: Comparison of foetal heart rate recorded by direct (scalp) and abdominal (FEMO) electrodes

Materials and methods 90

Figure 6-5b: Comparison of foetal ECG complex recorded by direct (scalp) and abdominal (FEMO) electrodes

2.3 Measurement of foetal ECG parameters

Figure 6-6 shows an example of the raw abdominal ECG strip comprising of

both maternal and foetal ECG complexes, the online display of foetal and maternal

heart rates, as well as the average foetal ECG complex, indicating the onset and

termination points used in the measurement of the various cardiac time intervals

These points were visually determined as the earliest observed deflection from the

isoelectric line (onset) and the latest observed return of the ECG signal to the

isoelectric line (termination) The durations of the cardiac intervals were then

determined from the averaged fECG signal

For analysis, each 10-minute recording of fECG was divided into 4 equal

periods of about 2.5 minutes (approximately >300 beats) whereby an average fECG

complex was generated for each period From each averaged fECG complex, cardiac

time intervals of the P wave duration (Po to Pt), PR interval (Po to Qo), QRS complex

(Qo to St), QT interval (Qo to Tt), and T wave duration (To to Tt) were determined

Each fECG recording yielded 4 sets of the above time intervals, from which their

mean values were calculated Since the QT interval is known to be dependent on the

heart rate, the QT intervals were corrected for heart rate (QTc) according to Bazett’s

formula (QTc = QT/√RR)

In a separate cohort of 197 foetuses, intrapartum fECG was performed in the

labour ward during the 1st stage of labour The recording equipment and protocol

were the same as those used in the antenatal fECG recordings except that the fECG

Materials and methods 92

(a) Raw abdominal ECG

Figure 6-6: (a) Raw abdominal ECG strip, (b) foetal and maternal heart rate

traces, and (c) averaged foetal ECG complex recorded by FEMO

M= maternal QRS complex; F= foetal QRS complex;

Subscripts o and t = onset and termination points of P, QRS and T waves

recording was performed only once (during labour) on each patient Similar to

antenatal fECG, each recording of intrapartum fECG was performed for 10 minutes

In addition to the above cardiac time intervals, the T/QRS ratio and conduction index

were also computed for this group of foetuses T/QRS ratio was calculated by

dividing the T wave amplitude by the QRS peak-to-peak amplitude whereas the

conduction index was computed as the Pearson’s correlation coefficient of the PR

interval with the fHR

2.4 Neonatal ECG acquisition and measurement

For the mothers who gave written consent, a standard resting 12-lead ECG

was recorded for each neonate one to two days post-partum using ECG equipment

MAC5000 (Marquette GE Medical Systems Information Technologies, Milwaukee,

Wisconsin, USA) The ECG was recorded with the neonate in a supine resting

position, using a paper speed of 25 mm/s The neonatal ECG parameters measured by

MAC5000 were the durations of PR interval, QRS interval, QT interval and QTc

interval

The reason for measuring neonatal ECG was to compare the

electrocardiographic changes in the ECG before and after birth Nevertheless it is to

be noted that equipment used for recording foetal and neonatal ECG were different

Intra- and extra-uterine conditions were also not similar

Materials and methods 94

2.5 Foetal HRV measurement

For analysis of foetal HRV, a computer system (F-EXTRACT) was developed

in collaboration with the Computer Science Department of National University of

Singapore This HRV program was developed using MatLab 6.1 (Release 12.1) and

requires MatLab installation to run A more detailed description of F-EXTRACT is

discussed in Chapter 9

From the foetal ECG signals, the RR-intervals were extracted for HRV

analysis Using F-EXTRACT, foetal HRV was determined in both time and

frequency domains The time domain indices measured were the mean foetal heart

rate, mean RR interval, SDNN, rMSSD and pNN27 Mean heart rate is the mean

foetal heart rate measured in beats per minute The mean RR interval is the average

duration of all the RR intervals during the recording It is also known as mean NN

interval (mNN), indicating normal-to-normal intervals, i.e., between beats in sinus

rhythm

SDNN is the standard deviation of NN interval and is defined as:

where NN i is the duration of the i-th NN interval, n is the number of all NN intervals

and m is their mean duration Since variance (square of standard deviation) is

mathematically equal to total power of spectral analysis,SDNN reflects total HRV,

i.e., all the cyclic components responsible for heart rate variation during the period of

recording, such as respiratory, baroreceptor, thermoregulation, etc

Another time domain index measured is the rMSSD, which is the root mean

square of successive differences between adjacent NN intervals, mathematically

defined as:

where NN i is the duration of the i-th NN interval and n is the number of all NN

intervals

Finally, pNN27 is another time domain index measured in this study It is

defined as the percentage of the number of pairs of adjacent NN intervals differing by

more than 27 ms in the entire recording divided by the total number of all NN

intervals This index is a modified version of the more familiar pNN50, which is

defined as the percentage of the number of pairs of adjacent NN intervals differing by

more than 50 ms in the entire recording (NN50) divided by the total number of all NN

intervals The absolute threshold of 50 ms in the pNN50 index makes it dependent on

the underlying heart rate A lower heart rate (longer RR intervals) has a higher chance

of adjacent intervals differing by more than 50 ms than a higher heart rate (shorter RR

intervals) Hence a proportional threshold of 6.25% of the mean RR interval has been

proposed (Mietus JE et al., 2002; Malik M, 1997; Ewing DJ et al., 1984) This

corresponds to a 50 ms difference at the adult heart rate of 75 beats per minute (bpm)

However, the average heart rate of the fetus is almost twice that of the adult’s, with

the average being 140 bpm Thus, using the threshold of 6.25%, a pNN27 would be

more suitable for performing HRV calculation in the fetus

Materials and methods 96

As for HRV analysis in the frequency-domain, the first step was to produce a

foetal tachogram for each fECG recording, whereby mean NN intervals were plotted

against their beat number Next, a 256-second of artifact-free segment was selected

from the tachogram for generating the foetal HRV power spectrum, which was based

on the Fast Fourier Transformation (FFT) The area under each power spectrum was

then calculated for three regions: very low frequency (VLF: 0.003-0.04 Hz), low

frequency (LF: 0.04-0.15 Hz) and high frequency (HF: 0.15-1.0 Hz) Other calculated

frequency-domain parameters included the total spectral power, normalized LF and

HF power, as well as LF/HF ratio Total power refers to the total power in the HRV

spectrogram The LF/HF ratio was computed by dividing the HF power by LF power,

while normalized LF and HF power were calculated by the following formulae:

Normalized LF = LF/(TP-VLF) x100

Normalized HF = HF/(TP-VLF) x100

2.6 Comparison of F-EXTRACT and Nevrokard HRV softwares

The HRV measurements derived from the F-EXTRACT was compared to

those derived from a commercial HRV software, the Nevrokard HRV System

(Medistar Inc., Slovenia) Both HRV systems were utilized to perform the same set of

calculations on the same time epochs of foetal RR-interval samples Bland-Altman

analysis (Bland JM and Altman DG, 2003) was used to evaluate the level of

agreement between the two programs

2.7 Correction of aberrant beats

Since HRV is derived from the measurement of heart cycle period or R-R

interval, a missing beat will result in a longer than normal R-R interval Similarly, an

ectopic beat (often premature) will produce a short R-R interval followed by a

compensatory delay, and hence a prolonged interval For this reason, ectopic or

missing beats introduce significant errors in the HRV statistics and distort the HRV

spectrum, thereby making it difficult to evaluate a patient’s neurocardiac control

through HRV techniques (Berntson GG et al., 1998) Therefore, these aberrant beats

must be excluded from the HRV calculation F-EXTRACT enables the automatic

detection and exclusion of any abnormal beats or artifacts before running FFT to

construct the power spectral plots QRS complexes classified as noise or ectopic beats

were excluded Only normal-to-normal intervals (in sinus rhythm) were used in the

calculation of HRV

2.8 Statistics

Statistical analyses were performed using SPSS 12.0 for Windows (SPSS Inc.,

Chicago, IL, USA) The results were expressed as mean ± SD Statistical significance

was assumed at a level of p < 0.05 Statistical analyses of independent samples t-tests,

one-way ANOVA and linear regression were performed using SPSS 12.0 for

Windows, while Bland-Altman analyses were performed using Prism 4.03 for

Windows (GraphPad Software Inc., San Diego, CA, USA)

ECG of healthy foetuses 98

CHAPTER 7 CARDIAC TIME INTERVALS

OF HEALTHY FOETUSES

1 Introduction

As in the adult, the foetal electrocardiogram (fECG) provides important information about the foetal cardiac electrical activity Foetal cardiac time intervals may be useful for the diagnosis of arrhythmias (Abe K et al., 2005; Sturm R

et al., 2004; Yumoto Y et al., 2004), heart blocks (Lin MT et al., 1998; Mohajer MP

et al., 1995), long QT syndrome, (Schneider U et al., 2005; Hosono T et al., 2002; Menéndez T et al., 2000; Hamada H et al., 1999), intra-uterine growth restriction (IUGR) (Grimm B et al., 2003; van Leeuwen P et al., 2001; Pardi G et al., 1986) and foetal hypoxaemia (Rosen KG, 2005; Reed NN et al., 1996; Mohajer MP et al., 1994)

In 1986, Murray (Murray HG, 1986) observed that the conduction index (Pearson’s correlation coefficient of the PR interval with foetal heart rate (fHR)), which was normally negative, became positive during foetal acidosis Subsequent studies demonstrated the effectiveness of the use of conduction index in the reduction

of foetal acidosis as well as unnecessary foetal blood sampling and instrumental deliveries (Reed NN et al., 1996; van Wijngaarden WJ et al., 1996b)

Other fECG studies showed that the ST waveform reflects the metabolic events occurring at a tissue level in response to compensatory mechanisms for oxygen lack in a vital central organ A progressive rise of the foetal ST waveform quantified

by T/QRS ratio indicates prolonged myocardial stress and a switch to anaerobic metabolism (Rosen KG and Luzietti R, 1994; Rosen KG and Lindecrantz K, 1989;

ECG of healthy foetuses 100

Jenkins et al., 1986; Rosen KG, 1986a; Lilja et al., 1985; Rosen KG et al, 1976; Rosen KG et al., 1975)

Based on these findings, the STAN foetal monitor (ST Analyser, Cinventa

AB, Sweden) has been developed to evaluate intrapartum fECG in terms of the ST segment and the T wave using foetal scalp electrodes The addition of ST monitoring

to standard fHR monitoring has shown to be useful in predicting fetal acidosis Wahlin I et al., 2002), improving the clinical decision making for obstetric interventions (Amer-Wahlin I et al., 2005; Kwee A et al., 2004; Ross MG et al., 2004), thereby improving perinatal outcome (Rosen KG, 2005; Olofsson P, 2003)

(Amer-Instead of using scalp electrodes, fECG can be non-invasively obtained using electrodes placed on the maternal abdomen However, despite the usefulness of fECG and the observations of configuration changes in abdominal fECG in relation to foetal hypoxia, problems of electrical noise and signal distortion have restricted its application for routine clinical monitoring of hypoxia in the human foetus

The main problem in measuring abdominal fECG is the poor signal-to-noise ratio Foetal ECG is often superimposed and hidden by maternal ECG (mECG), which is many times higher in intensity than fECG (Peters M et al., 2001) Maternal electromuscular activity, external electrical interferences (50 Hz noise), and the intrinsic noise of the equipment also contribute to the low signal-to-noise ratio In order to obtain clear abdominally-derived fECG from which diagnostic parameters

can be derived, the main aim is to remove the various contributions to noise that may mask the low fECG signal

Advances in signal recognition and separation now allows fECG to be isolated from the mECG Sophisticated filtering, processing and amplification techniques are used to yield clear fECG complexes containing P, QRS and T waves The aim of this research is to utilize a new equipment, FEMO (Medco Electronic Systems Ltd., Israel), which uses a non-invasive abdominal technique to measure foetal cardiac time intervals in normal singleton pregnancies

In order to discriminate between normal and pathological changes in the fECG, a database of normal values of cardiac time intervals for reference is essential Serial fECGs from a cohort of 100 healthy foetuses were measured from 18 to 41 weeks of gestation To the best of my knowledge, no known longitudinal follow-up studies of fECG cardiac time intervals obtained from abdominal fECG have been described in the literature The data in this study will contribute to the understanding

of normal cardiac time intervals in foetuses at various gestational ages

2 Study population

The study population consisted of 100 women with singleton pregnancies with

no clinical maternal or foetal complications The women were aged between 22 and

41 years (mean ± SD: 31.8 ± 3.8 years) The foetuses were divided into 5 groups

ECG of healthy foetuses 102

according to gestational age (GA) These groups were: 18-22, >22-27, >27-32,

>32-<37, and ≥ 37 weeks

The basis of dividing the foetuses into the various GA is as follows: the first group of 18-22 weeks describes the non-viable age for foetuses (Macfarlane PI et al., 2003; Allen MC et al., 1993; Nicholl MC et al., 1991) Sometimes called “micro-premies”, foetuses born during >22-27 weeks are regarded as extremely preterm The early neonatal mortality is high, with up to 50% of severe disability occurring in those

born before 26 weeks (Molholm HB et al., 2002; Wood NS et al., 2000) Their

chances of survival vary significantly depending on multiple factors such as foetal age, weight and gender, steroid/surfactant treatment, mode of presentation, multiple

pregnancy, etc (Effer SB et al., 2002; El-Metwally D et al., 2000; Battin M et al., 1998; Kramer WB et al., 1997; Lefebvre F et al., 1996; Whyte HE et al., 1993)

According to the World Health Organization, an infant born before the 37th week of gestation is defined as preterm (WHO, 1993) Hence the 5th group of foetuses aged ≥ 37 weeks constitutes term foetuses Moderate prematurity refers to foetuses of

>32-<37 weeks of gestational age Foetuses born between >27-32 weeks are considered as very preterm (Surkan PJ et al., 2004; Moutquin JM et al., 2003)

3 Method

Foetal ECG recording was performed on all the 100 foetuses for 10 minutes during each visit Average cardiac time intervals of the P wave duration, PR interval, QRS complex, QT interval, QTc interval and T wave duration were determined

Intrapartum fECG recording was performed on a separate cohort of 197 foetuses during the 1st stage of labour The recording protocol was similar to that used for antenatal fECG Measurements included the above cardiac time intervals, as well

as the T/QRS ratio and conduction index

Neonatal ECG recording was performed on a subset (n=51) of the cohort of

100 foetuses at 1-2 days after delivery using a standard resting 12-lead ECG The cardiac time intervals obtained from the neonatal ECG results include the PR interval, QRS duration, QT interval and QTc interval A detailed description of the above methods can be found in Chapter 6

4 Results

4.1 Success rates of foetal ECG recording

The success rates of detecting the various cardiac waveforms on the fECG are displayed in Table 7-1 On the whole, the QRS complex was successfully measured

in 92.1% of the recordings, while P and T waves were recognized in 76.8% and 81.0% of all recordings, respectively The PR and QT intervals had success rates similar to P and T waves, respectively Success rates were generally poorer during the