A Comparison of the Inhibitory Effects of Bupivacaine

and Levobupivacaine on Isolated Human Pregnant

Myometrium Contractility
Rebecca A. Fanning, FCARCSI*
Deirdre P. Campion, PhD†
Colm B. Collins, PhD†
Simon Keely, PhD†
Liam P. Briggs, FFARCSI*
John J. O’Connor, PhD‡
Michael F. Carey, MD*
BACKGROUND: Epidural analgesia with levobupivacaine and bupivacaine is a com-
mon and effective method of labor pain relief. However, its use is associated with
an increased instrumental delivery rate. One of the mechanisms postulated to
account for this unwanted effect is the direct effect of local anesthetics on
myometrial contractility. We determined the effects of bupivacaine and levobupiva-
caine on the amplitude and frequency of contractions of human term myometrium.
METHODS: Uterine specimens were obtained from nonlaboring parturients sched-
uled for elective lower-segment cesarean delivery at term. Longitudinal muscle
strips were prepared and mounted vertically in tissue chambers, and changes in the
amplitude (peak force) and the frequency of contractions were recorded. Sponta-
neous contractions commenced after a period of application of 1 g (9.81 mN) of
tension to the myometrial strips. No uterotonic drugs were used. The muscle strips
were then exposed to cumulative concentrations of bupivacaine and levobupiva-
caine and dose–response curves were generated.
RESULTS: Both bupivacaine and levobupivacaine decreased the amplitude of con-
tractions in human myometrium in a concentration-dependent manner, reaching
significance at 1 ⫻ 10⫺4 M for both bupivacaine and levobupivacaine compared
with the internal control amplitude. With both drugs, the decrease in amplitude
was accompanied by an increase in the frequency of contractions reaching
significance at 3 ⫻ 10⫺5 M for both bupivacaine and levobupivacaine compared
with the internal control frequency.
CONCLUSIONS: The concentrations required for the effects on amplitude are much
higher (33 fold) than the clinically relevant plasma concentrations of these drugs
after epidural administration, and are unlikely to be significant in the setting of
low-dose epidural analgesia in labor.
(Anesth Analg 2008;107:1303–7)

E pidural analgesia is a mode of pain relief com-

monly used for labor pain (35%– 61% United States1
and 22.5% United Kingdom2). Although the mecha-
nisms responsible for the initiation and progress of labor
are still not well understood,3 the effects of epidural
analgesia on the progress and outcome of labor have
been the focus of a large amount of research in the last
decade. Relative to other forms of labor pain relief,
epidural analgesia is the most effective but is associated
with increased instrumental delivery rate,4 – 6 longer sec-
ond stage of labor,4 –7 and increased rates of oxytocin
augmentation.4 –7 One of the mechanisms postulated to
From the *Department of Perioperative Medicine, Coombe
Women and Infants University Hospital, †University College Dub-
lin School of Agriculture, Food Science and Vetenary Medicine, and
‡Conway Institute of Biomolecular and Biomedical Science, Univer-
sity College Dublin, Dublin, Ireland.
Accepted for publication May 1, 2008.
Address correspondence and reprint requests to Dr. Rebecca
Fanning, Department of Perioperative Medicine, Coombe Women
and Infants University Hospital, Dublin 8, Ireland. Address e-mail
to rebecca.fanning@gmail.com.
Copyright © 2008 International Anesthesia Research Society
DOI: 10.1213/ane.0b013e3181804245
account for these epidural analgesia-related adverse ef-
fects arises from the direct effects of local anesthetics on
the propagation and strength of myometrial contrac-
tions.8 Local anesthetics routinely used in obstetric anes-
thesia practice include bupivacaine and its enantiomer
levobupivacaine. Levobupivacaine has a clinical profile
closely resembling that of bupivacaine, but is less toxic to
both the central nervous and cardiovascular systems.9
These attributes make this drug particularly appealing
for use in the setting of obstetric anesthesia, where large
volumes of local anesthetic may be required. In vitro
findings have suggested that these drugs demonstrate
differential effects in their ability to block cardiac sodium
and potassium channels,10 which raises the possibility of
differential effects in myometrial tissue. Previous studies
have reported that bupivacaine11–13 and levobupiva-
caine13 cause concentration-dependent inhibition of the
amplitude of gravid rat myometrial contractions. The
effects of bupivacaine and levobupivacaine have not been
reported in human myometrium in the in vitro setting. We
hypothesized that bupivacaine and levobupivacaine may
have differential effects on the amplitude and frequency of
human myometrial contractility.

Vol. 107, No. 4, October 2008 1303

METHODS
Tissue Sample Preparation
The protocol for the study was approved by the
local IRB. After obtaining written informed consent, 10
term ASA physical status 1 pregnant women sched-
uled for elective lower segment cesarean delivery
were enrolled. None of the women were in labor. All
patients received spinal anesthesia with 10 –12 mg
0.5% hyperbaric bupivacaine, with 20 –25 ␮g of intra-
thecal fentanyl, and 100 –150 ␮g of intrathecal mor-
phine. After delivery, all women received 5 IU of
oxytocin IV (Novartis Pharmaceuticals UK Ltd., Hor-
sham, West Sussex, UK) After the placenta was deliv-
ered, a small segment of myometrium was excised
from the upper incisional surface of the lower uterine
segment. The specimen was washed and stored in
lactated Ringer’s solution, and refrigerated at 4°C
until the experiment began 2–18 h later.
Contractility Experiments
Each specimen was dissected into at least four
longitudinal muscle strips 12 mm long, 5 mm wide,
and 1 mm thick. The strips were mounted vertically
into individual 10- or 15-mL organ baths (Myobath,
World Precision Instruments Inc., Sarasota, FL) with
Krebs solution and connected to individual force
transducers (Transbridge 4M, World Precision Instru-
ments Inc.) to record isometric tension. The Krebs–
Henseleit bicarbonate buffer solution contained: NaCl
118 mmol/L, d-glucose 11.1 mmol/L, NaHCO3 24.9
mmol/L, MgSO4 1.2 mmol/L, KCl 4.7 mmol/L,
KH2PO4 1.2 mmol/L, and CaCl2 2.5 mmol/L. The
organ baths were aerated with a gas mixture of 95%
oxygen and 5% CO2 and maintained at 36.7°C. A
resting tension of 1g (9.81 mN) (based on protocols
from similar experimental work11,12) was initially ap-
plied, and subsequently reapplied as necessary over the
first 30 – 40 min until a steady tension was achieved.
Simultaneously, the Krebs solution was changed every
10 min for 30 min to remove any residual oxytocin. At no
time were contractions stimulated with oxytocin or other
uterotonic drugs. Spontaneous contractions usually de-
veloped over the following 60 –90 min. When the con-
tractions became regular in amplitude and frequency,
and after a 30-min control period, the muscle strips
were randomly exposed to either bupivacaine (Astra-
zeneca, UK) or levobupivacaine (Abbott Laboratories,
USA). Drug-containing solutions were prepared
immediately before each experiment using Krebs so-
lution. The strips were exposed to cumulative concen-
trations of 10⫺8 to 3 ⫻ 10⫺4 M for each local anesthetic.
There was a 30-min interval between each addition
and drugs were added in the following increments:
1 ⫻ 10⫺8 M, 1 ⫻ 10⫺7 M, 3 ⫻ 10⫺7 M, 1 ⫻ 10⫺6 M, 3 ⫻
10⫺6 M, 1 ⫻ 10⫺5 M, 3 ⫻ 10⫺5 M, 1 ⫻ 10⫺4 M, and 3 ⫻
10⫺4 M. A control muscle strip was maintained for
each sample to which no drugs were added to ensure
viability for the duration of the experiment. After the
addition of the 3 ⫻ 10⫺4 M concentration of each local
1304 Effects of Bupivacaine and Levobupivacaine on Human Pregnant Myometrium
anesthetic, the baths were washed out every 10 min
with warmed Krebs solution for a period of 30 min to
ascertain if the effect of the local anesthetics could be
reversed. After completion of the experiment, the
weight (0.089 ⫾ 0.01 g) and dimensions of each muscle
strip were recorded.
The amplitude (peak force) and frequency of con-
tractions were recorded for each drug treatment
window and subsequently analyzed using Powerlab
Program (AD Instruments Pty Ltd., Australia). The
amplitude of contraction is expressed as a % of
the amplitude measured in the control period before the
addition of drug. The frequency of contractions was
assessed by measuring the peak-to-peak interval, in
seconds, between the last two contraction peaks for each
drug window and expressed as a percent of the fre-
quency measured in the control period. The amplitude
and frequency in the control period were taken as 100%.
Data Analysis
Data are presented as mean ⫾ sd. Statistical analysis
was performed using GraphPad Prism 4 statistical soft-
ware (GraphPad Software Inc., USA). Dose–response
curves and log EC50 values were generated using non-
linear regression analysis. The effects of levobupivacaine
and bupivicaine on contractility were compared using
one-way repeated measures analysis of variance. A
two-tailed paired t-test was used to compare the effects
of increasing bupivacaine and levobupivacaine concen-
trations to the internal control period. P ⬍ 0.05 was
considered to be statistically significant.
RESULTS
Eight of the 10 control strips of muscle (to which no
drugs were added) contracted without fade for the
duration of the experiment (6 –7 h). The other two
failed to contract and so myometrial strips from these
women were excluded. Figure 1 depicts a typical
tracing generated by the spontaneous contractions of
an isolated myometrial strip, showing a reduction in
the amplitude and interval between contractions with
increasing concentrations of bupivacaine, and revers-
ibility after drug washout at the end. The mean
tension that developed during the control period (n ⫽
8, where n ⫽ the number of patients) was 8.61 ⫾
4.71 g, with a mean peak-to-peak interval of 627 ⫾ 65 s
and a contraction rate of 6.9 ⫾ 0.1 contractions per
hour.
Local anesthetic effects: There were eight strips of
muscle in each of the bupivacaine, levobupivacaine,
and control groups. Bupivacaine and levobupivacaine
caused a concentration-dependent decrease in the
amplitude of contractions (Fig. 2) reaching statistical
significance at 1 ⫻ 10⫺4 M (P ⫽ 0.002 for levobupiva-
caine and P ⫽ 0.01 for bupivacaine) compared with
the control period before the addition of any drugs.
This decrease in amplitude of contractions was accom-
panied by a decrease in peak-to-peak interval, indicat-
ing an increased frequency of contractions (Fig. 3).
ANESTHESIA & ANALGESIA
Figure 1. A sample tracing showing the effects
of increasing concentrations of bupivacaine on
the spontaneous contractions of isolated hu-
man term myometrium.
Figure 2. Effects of bupivacaine versus levobupivacaine on
amplitude of myometrial contractions (n ⫽ 8). Each point
represents the mean ⫾ sd. Comparison between bupiva-
caine and levobupivacaine was made using one-way re-
peated measures ANOVA. There was no difference found.
The effect of increasing concentrations of bupivacaine and
levobupivacaine compared to the internal control period
was analyzed with a two-tailed paired t-test *P ⬍ 0.05.
This decrease in peak-to-peak interval differed signifi-
cantly from the control period at 3 ⫻ 10⫺5 M for both
drugs (P ⫽ 0.02 for bupivacaine and P ⫽ 0.02 for
levobupivacaine). There were no significant differ-
ences between bupivacaine and levobupivacaine in
their effects on myometrial contractility. Table 1
shows the EC50 values (effective concentration which
causes 50% of the maximal effect) for amplitude of
contractions and interval between contractions for
bupivacaine and levobupivacaine. There were no dif-
ferences between the log EC50 values for these two
drugs. The effects of both drugs on contractility were
reversible.
DISCUSSION
Epidural analgesia remains the “gold standard” for
providing optimal pain relief in labor. However, its
use is associated with increased instrumental delivery
rates.4 – 6 Techniques that use low compared with
higher concentration local anesthetic solutions can
reduce this rate.14 This implies that local anesthetics
may be at least partly responsible for this unwanted
effect.
Vol. 107, No. 4, October 2008
Figure 3. Effects of bupivacaine versus levobupivacaine on
the interval between myometrial contractions (n ⫽ 8). Each
point represents the mean ⫾ sd. Comparison between
bupivacaine and levobupivacaine was made using one-way
repeated measures ANOVA. There was no difference found.
The effect of increasing concentrations of bupivacaine and
levobupivacaine compared with the internal control period
was analyzed with a two-tailed paired t-test *P ⬍ 0.05.
Table 1. Calculated log EC50 (Effective Concentration of
Drug that Produced a 50% Response) and the 95%
Confidence Intervals
Log EC50 (M) 95% CI
Amplitude
Bupivacaine ⫺4.21 ⫺4.42 to ⫺4.0
Levobupivacaine ⫺3.98 ⫺4.2 to ⫺3.76
P–P interval
Bupivacaine ⫺3.68 ⫺4.44 to ⫺2.92
Levobupivacaine ⫺5.14 ⫺5.88 to ⫺4.39
Log EC50 values were generated using nonlinear regression analysis (n ⫽ 8). There was no
difference between bupivacaine and levobupivacaine in the effect of drug concentration on
amplitude or peak-to-peak interval (P–P interval) of myometrial contractions.
There have been many reasons postulated to ex-
plain this effect including (1) the direct effect of local
anesthetics on myometrial contractility, (2) the motor
block, which may follow epidural analgesia leading to
decreased voluntary maternal expulsive efforts during
the second stage of labor, (3) paralysis of the pelvic
floor muscles that may retard or eliminate the descent
and rotation of the fetal head, and (4) attenuation of
the normal increase in oxytocin through inhibition of
the Ferguson (uteropituitary) reflex.15 We sought to
© 2008 International Anesthesia Research Society 1305
determine if two commonly used local anesthetics in
obstetric anesthesia had any direct effect on myome-
trial contractility in the in vitro setting. We found that
both bupivacaine and levobupivacaine caused a
concentration-dependent decrease in the amplitude of
contractions of term human myometrium. Both drugs
caused an increase in frequency of contractions.
The findings of the effects of bupivacaine on am-
plitude are in accord with previous studies of rat
myometrium.11–13 In contrast, Li et al. examined the in
vitro effects of levobupivacaine on rat myometrial
contractility13 and observed a biphasic effect with a
significant increase in amplitude of contractions from
3 ⫻ 10⫺7 M to 3 ⫻ 10⫺6 M, followed by a significant
reduction in amplitude of contractions at 1 ⫻ 10⫺4 M.
Our findings differ from these in that we observed no
increase in contractile amplitude. Many other studies
have demonstrated that bupivacaine has an inhibitory
effect on contraction in other types of smooth muscle,
e.g., canine papillary muscle,16 bladder smooth
muscle,17 isolated arterial smooth muscle,18 and tra-
cheal smooth muscle.19
The results of studies of the in vitro effect of local
anesthetics on the frequency of myometrial contrac-
tions are conflicting. Li et al.13 demonstrated bupiva-
caine had a biphasic effect increasing contractile
frequency at 3 ⫻ 10⫺5 M, whereas both bupivacaine
and levobupivacaine showed inhibitory, though non-
statistically significant, effects at 1 ⫻ 10⫺4 M concen-
tration. Other studies have demonstrated conflicting
results (an increased contractile frequency12 or no
effect on frequency11) on the effects of local anesthetics
on frequency of contractions. These differences may
be explained by the differences in human and animal
myometrium.20 The increase in frequency observed in
the current study may be related to altered intracellu-
lar calcium content resulting from the reduction in
contractile amplitude. In contrast to findings in myo-
cardial tissue, there was no difference observed between
bupivacaine and levobupivacaine in their ability to re-
duce myometrial contractility.10 Local anesthetics are
amphiphilic, and can enter a variety of cell compart-
ments and potentially interact with many different cell
membranes, organelles (including inhibition of mito-
chondrial adenosine triphosphate production21), and a
variety of both membrane-bound and cytosolic charged
molecules. Other mechanisms, which may account for
the myometrial inhibitory effect, include blocking of iono-
tropic signaling pathways (sodium, potassium, or cal-
cium) and interference with G-protein modulation of
calcium or potassium channels.22
Do the inhibitory effects of these local anesthetics
account for some of the unwanted side effects of labor
epidural analgesia? There is only one published
study23 reporting plasma levels of bupivacaine and
levobupivacaine in parturients after epidural admin-
istration. In this study, women received a bolus of 30
mL of 0.5% levobupivacaine or bupivacaine for cesar-
ean delivery anesthesia (150 mg). The maximal plasma
1306 Effects of Bupivacaine and Levobupivacaine on Human Pregnant Myometrium
concentrations were 1.053 ␮g/mL (3.24 ⫻ 10⫺6 M) for
bupivacaine and 1.017 ␮g/mL (3.13 ⫻ 10⫺6 M) for
levobupivacaine.23 We observed significant inhibitory
effects of bupivacaine and levobupivacaine on con-
tractile amplitude at concentrations which were 33
times higher (1 ⫻ 10⫺4 M) than this and 17 times
higher than the level at which central nervous system
toxicity has been reported to occur (8.07 ⫻ 10⫺6 M for
levobupivacaine and 6.92 ⫻ 10⫺6 M for bupiva-
caine24). The current practice of using low concentra-
tion (0.125%– 0.25%) solutions of local anesthetics
makes it unlikely that the inhibitory plasma concen-
tration of these drug levels would be reached in
clinical practice.
This study is limited by the same “experimental
noise” that is encountered in all studies of human
myometrial contractility; that is, that the myometrial
samples were obtained from women undergoing elec-
tive cesarean delivery at term. It is not known whether
the myometrium has undergone the complex molecu-
lar changes required for the onset and establishment
of labor at this stage.20 Samples obtained were from
the upper incisional surface of the lower uterine
segment of the uterus, the only tissue available in this
setting. However, regional differences to drugs have
been demonstrated in both animal25 and nonpregnant
human myometrium,26 and it is possible that different
effects may be observed in fundal myometrium. To
ensure tissue viability for the duration of the experi-
ment, we limited the exposure of the specimen to each
concentration of local anesthetic for 30 min (similar to
the protocol used in previously published similar
work11). We cannot be certain that the maximal effects
of that drug concentration can be observed in that
period. In addition, the interaction between the local
anesthetics and oxytocin that would occur in normal
labor was not examined. Despite three washouts with
Krebs solution, we also cannot be certain that some
residual oxytocin remained in the myometrial strips.
Despite these difficulties, it is important to understand
that there are differences between human and animal
myometrium, and studies such as this improve our
understanding of human myometrial function.
In summary, bupivacaine and its enantiomer levo-
bupivacaine, cause a similar concentration-dependent de-
crease in human myometrial contractile amplitude in the in
vitro setting. This is accompanied by an increase in the
frequency of contractions. The concentrations required
for the inhibitory effect on the amplitude of contractions
is much higher (33 fold) than the clinically relevant
plasma concentrations of these drugs after epidural
administration and are unlikely to be significant in the
setting of low-dose epidural analgesia in labor.
Therefore, a direct effect of local anesthetics on
uterine contractility is unlikely to explain the associa-
tion between epidural labor analgesia and instrumen-
tal vaginal delivery.
ANESTHESIA & ANALGESIA
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© 2008 International Anesthesia Research Society 1307