American Journal of Obstetrics and Gynecology (2006) 195, 504–9
www.ajog.org
Inhibitory effect of leptin on human uterine
contractility in vitro
Audrey T. Moynihan, MSc,a,b Mark P. Hehir,a Siobhan V. Glavey,a Terry J. Smith, PhD,b
John J. Morrison, MDa,b,*
Department of Obstetrics and Gynaecology,a Clinical Science Institute, University College Hospital Galway;
National Centre for Biomedical Engineering Science,b National University of Ireland, Galway, Ireland
Received for publication October 25, 2005; revised January 20, 2006; accepted January 27, 2006
KEY WORDS
Leptin
Myometrium
Maternal obesity
Parturition
Objective: The purpose of this study was to investigate the effects of leptin on human uterine
contractility in vitro.
Study design: Biopsies of human myometrium were obtained at elective cesarean section (n =
18). Dissected myometrial strips suspended under isometric conditions, undergoing spontaneous
and oxytocin-induced contractions, were exposed to cumulative additions of leptin in the concentration range of 1 nmol/L to 1 mmol/L. Control strips were run simultaneously. Integrals of contractile activity were measured using the PowerLab hardware unit and Chart v3.6 software.
Results: Leptin exerted a potent and cumulative inhibitory effect on spontaneous and oxytocininduced contractions compared to control strips. The mean maximal inhibition values were as
follows: 46.794 G 5.133% (n = 6; P ! .001) for spontaneous contractions and 42.323 G
3.692% (n = 6; P ! .001) for oxytocin-induced contractions. There was an apparent reduction
in both frequency and amplitude of contractions.
Conclusion: This physiologic inhibitory effect of leptin on uterine contractility may play a role in
the dysfunctional labor process associated with maternal obesity, and the resultant high cesarean
section rates.
Ó 2006 Mosby, Inc. All rights reserved.
Obesity is rapidly becoming a major health problem
in the developed world. Apart from the general adverse
health implications of obesity, it is now apparent that
obesity in pregnancy is linked to a significant increase
Support from Higher Education Authortiy (HEA) of Ireland
PTRLI3.
* Reprint requests: Professor John J. Morrison, MD, MRCOG,
FRCPI, BSc, DCH, Department of Obstetrics and Gynaecology, Clinical Science Institute, University College Hospital Galway, Newcastle
Road, Galway, Ireland.
E-mail: [email protected]
0002-9378/$ - see front matter Ó 2006 Mosby, Inc. All rights reserved.
doi:10.1016/j.ajog.2006.01.106
in complications such as failure of the labor process,
cesarean section, hypertensive disorders, gestational
diabetes mellitus, and fetal macrosomia.1,2 There are
numerous possible explanations for the association between obesity and failure of normal parturition, which
include increased fat deposition in the maternal pelvis,
fetal macrosomia, and an inadequate active second
stage of labor. The possibility of altered metabolic
modulation of myometrial smooth muscle in association with obesity has hitherto not been considered,
despite the widely reported functional roles of secretory products of adipose tissue, and the emerging
Moynihan et al
concept that these secretory products play an important role in the pathophysiology of obesity related
complications.3
Leptin is a peptide with 167 amino acid residues that
is secreted from adipose tissue under the control of the
obesity gene,4 and is known to have regulatory effects on
neuronal tissue, vascular smooth muscle, and nonvascular smooth muscle systems.5 Leptin concentrations rise
significantly with increasing percentage body fat,6 and
obese individuals have markedly increased leptin production.7 The role of leptin in human reproduction is
not clear. While leptin is most likely involved in the regulation of ovarian function, oocyte maturation, embryo
development and implantation,8 the role of leptin during
pregnancy has not been established. The placenta is a
major source of leptin production during pregnancy,9
and leptin may play a role in the pathophysiology of preeclampsia10 and hypertensive disorders of pregnancy.11
Leptin and leptin receptor genes are also expressed
in human umbilical cord, fetal membranes, and uterine
tissue.12
In the human,13 as well as in rat14 and higher mammalian models,15 serum leptin levels are markedly elevated during pregnancy and demonstrate a significant
decrease just before or at the time of parturition. Because of this, and because of the diverse metabolic functions associated with leptin, we hypothesized that leptin
may modulate myometrial function and activity. The
aim of this study was therefore to investigate the effects
of leptin on human uterine contractility in vitro, on
spontaneous contractions and those elicited by the agonist oxytocin.
Material and methods
Tissue collection
Biopsies of human myometrial tissue were obtained
at elective cesarean section in the third trimester of
pregnancy in the Department of Obstetrics and Gynecology, University College Hospital, Galway, Ireland.
The biopsies were excised from the upper lip of the
lower uterine segment incision in the midline, ie, upper
portion of lower uterine segment. Ethical committee
approval for tissue collection was obtained from the
Research Ethics Committee at University College Hospital Galway and recruitment was by written informed
consent. Once collected, all tissue biopsies were placed in
Krebs-Henseleit physiologic salt solution (PSS) at pH
7.4 containing the following: 4.7 mmol/L potassium
chloride, 118 mmol/L sodium chloride, 1.2 mmol/L
magnesium sulphate, 1.2 mmol/L calcium chloride, 1.2
mmol/L potassium phosphate, 25 mmol/L sodium bicarbonate, and 11 mmol/L glucose (Sigma-Aldrich,
Dublin, Ireland). Tissues were stored at 4(C and used
within 12 hours of collection.
505
Tissue bath experiments
Longitudinal myometrial strips (measuring approximately 2 ! 2 ! 10 mm) were dissected free of uterine
decidua and serosa and mounted for isometric recording
under 2 g of tension in organ baths as previously
described.16,17 The tissue baths contained 10 mL of
Krebs-Henseleit PSS maintained at 37(C, pH 7.4, and
were gassed continuously with a mixture of 95% oxygen/5% carbon dioxide. Myometrial strips were allowed
to equilibrate for at least 1 hour during which time the
Krebs-Henseleit physiologic salt solution was changed
every 20 minutes. After equilibration, a 30-minute period
was allowed in order to achieve spontaneous phasic contractions, or, alternatively, contractions were stimulated
by bath exposure of the strips to oxytocin (0.5 nmol/L).
Leptin was then added to the tissue bath in a cumulative
manner at bath concentrations of 1 nmol/L, 10 nmol/L,
100 nmol/L, and 1 mmol/L at 20-minute intervals. There
were 2 sets of control experiments performed as follows:
control 1. Strips exposed to either PSS only (for spontaneous contractions) or 0.5 nmol/L oxytocin; and control 2.
Strips exposed to PSS and vehicle for leptin (spontaneous
contractions) or 0.5 nmol/L oxytocin and vehicle for leptin. The effect of leptin and the respective controls were
assessed by calculation of the integral from minimum of
selected areas for each 20-minute intervals and expressed
as a percentage of the integral obtained in the 20-minute
period before any leptin addition using the PowerLab
hardware unit and Chart v3.6 software (AD Instruments,
Hastings, UK). The inhibitory effect of leptin was corrected for the reduction in the contractile activity observed in the vehicle control (control 2), and the effects
of leptin were interpreted as the final additional relaxant
effect. This value, when subtracted from 100% represents
the mean maximum inhibition (MMI) of leptin on myometrial contractility. The MMI provided therefore represents the net final inhibition resulting from exposure to
leptin, ie, after subtraction of any alteration observed in
vehicle control (control 2) experiments.
Drugs and solutions
Leptin was purchased from Sigma-Aldrich. A stock
solution (100 mmol/L) was made in 15 mmol/L hydrochloric acid and 7.5 mmol/L sodium hydroxide. Serial
dilutions were made in deionized water on the day of
experimentation. Fresh Krebs-Henseleit solution was
made daily. A stock solution of oxytocin (1 mmol/L;
Sigma-Aldrich) was prepared using deionized water.
Serial dilutions were prepared in deionized water on
the day of experimentation.
Statistical analysis
Comparisons of contractile effect, for each bath concentration of leptin, were performed using analysis of
506
Moynihan et al
variance (ANOVA) followed by Tukey HSD post hoc
testing to determine significant differences among data
groups. A P value of ! .001 was considered to be statistically significant. A paired sample t test was used to
compare spontaneous and oxytocin-induced vehicle controls to spontaneous and oxytocin-induced controls, respectively. The paired sample t test showed that there
was no significant difference between the spontaneous
vehicle control and the spontaneous control (P = .116)
and it showed that there was no significant difference
between the oxytocin-induced vehicle control and the
oxytocin induced control strips (P = .164). The statistical package SPSS for Windows version 11.0 (SPSS, Inc,
Chicago, IL) was used for these statistical calculations.
Results
Myometrial biopsies were obtained from a total of 18
women who underwent cesarean delivery between 38
and 40 weeks of gestation (median gestation, 38 weeks).
The reasons for cesarean section were previous cesarean
section (n = 15), placenta praevia grade 4 (n = 1),
breech presentation (n = 1), and previous pregnancy
losses (n = 1). The mean maternal age at delivery was
32.5 years (range 22-41 years). The median parity value
of the women at the time of delivery was 1 (range 0-3).
All cesarean sections were carried out under regional
anesthesia.
Leptin exerted an inhibitory effect on both spontaneous and oxytocin-induced contractions in human
myometrium in vitro, in all strips, in comparison to
control measurements. A representative recording of
spontaneous myometrial contractions is shown in
Figure 1A (control 1), and Figure 1B demonstrates a recording of spontaneous myometrial contractions treated
with increasing concentrations of vehicle for leptin (1
nmol/L, 10 nmol/L, 100 nmol/L, and 1 mmol/L) (control
2). Spontaneous controls 1 and 2 were compared using a
paired t test and no significant differences were found
between the 2 controls, P = .116. Figure 1C demonstrates the uterorelaxant effect of leptin on spontaneous
contractions in myometrial tissue with an observed
reduction in both amplitude and frequency. The results
of the calculated integrals of contractile activity are provided in the Table. A mean maximal inhibition (MMI)
of 46.794 G 5.133% (n = 6) was recorded (P !
.001). The reduction in contractility for spontaneous
contractions was cumulative and attained statistical significance at a bath leptin concentration of 1 mmol/L, in
comparison to control strips (with vehicle). This is demonstrated in Figure 2A, where a graphic representation
of the effect of increasing concentrations of leptin on
spontaneous myometrial contractions is shown.
The results obtained for oxytocin-induced myometrial contractility are similarly shown in Figure 3 and the
Figure 1 Effects of leptin on spontaneous myometrial contractility in pregnant nonlaboring tissue. Representative
recordings demonstrating A, spontaneous contractions in a
control strip, B, spontaneous contractions treated with vehicle
only, and C, the effects of cumulative additions of leptin
(1 nmol/L–1 mmol/L) in 20-minute intervals are shown.
Table. Figure 3A and B shows recordings of oxytocininduced myometrial contractions (control 1) and oxytocin-induced myometrial contractions treated with
increasing concentrations of vehicle (1 nmol/L, 10 nmol/L,
100 nmol/L, and 1 mmol/L) (control 2), respectively.
Oxytocin-induced controls 1 and 2 were compared using
a paired t test and no significant differences were found
between the 2 controls, P = .164. Figure 3C shows a representative recording of oxytocin induced myometrial
contractions treated with leptin and demonstrates the
uterorelaxant effect of leptin on contractility. A mean
maximal inhibition (MMI) of 42.323 G 3.642% (n = 6)
was recorded. The reduction in contractility for oxytocin-induced contractions was cumulative and attained
statistical significance at a bath leptin concentration of
1 nmol/L (P ! .001) and increasing concentrations thereafter of 10 nmol/L, (P ! .05), 100 nmol/L (P ! .001), and
1 mmol/L (P ! .05) in comparison to control strips (with
Moynihan et al
Table
507
Mean maximal inhibition (MMI) values produced by leptin on human myometrial contractility
Drug
Contraction Type
MMI (%) G SEM
P value
Leptin
Leptin
Spontaneous
Oxytocin-induced
46.794 G 5.133
42.323 G 3.642
n = 6, P ! .001*
n = 6, P ! .001*
Values are given for the net relaxatory effect (percentage value of the mean relaxatory effect that was observed in study strips minus the mean relaxatory
effect that was observed in vehicle control strips) of leptin. The values provided represent % inhibition G standard error of the mean (SEM). Values were
compared using ANOVA and Tukey post hoc test.
* P ! .001 versus control.
Figure 2 A, The dose response curves demonstrate the
uterorelaxant effect of cumulative increasing tissue bath
concentrations of leptin (1 nmol/L–1 mmol/L) on spontaneous-induced contractions (closed squares). A control trace
showing consistent spontaneous contractions treated with
vehicle only are shown for comparison (closed triangles). B,
The dose response curves demonstrate the uterorelaxant effect
of cumulative increasing tissue bath concentrations of leptin
(1 nmol/L–1 mmol/L) on oxytocin-induced contractions (closed
squares). A control trace showing uninterrupted oxytocininduced contractions treated with vehicle only is shown for
comparison. Percentage contractility is shown on the y-axis,
and the concentration of the relaxant is shown on the x-axis.
The points represent the means and the vertical error bars represent the standard error of the means. The asterisks denote
the concentrations at which the inhibitory effect of leptin
was statistically significant.
vehicle). This is demonstrated in Figure 2B, where a
graphic representation of the effect of increasing concentrations of leptin on oxytocin-induced myometrial contractions is shown.
Figure 3 Effects of leptin on oxytocin-induced myometrial
contractility in pregnant nonlaboring tissue. Representative recordings of A, oxytocin-induced myometrial contractions in
control strips, B, oxytocin induced contractions treated with
vehicle only, and C, the effects of cumulative additions of leptin (1 nmol/L–1 mmol/L) in 20-minute intervals are shown.
Comment
The results from this study demonstrate that leptin acts
as a potent inhibitor of myometrial contractility in vitro.
This relaxant effect was observed at relatively low
concentrations for in vitro experiments and was apparently cumulative in nature. Observing the raw data, it
was evident that leptin reduced both amplitude and
frequency, but for proper accuracy and analysis of
508
results, the integrals of contractile activity measured
over a period of time were compared. The findings from
these experiments are robust because of the fact that 2
separate series of control experiments were performed.
The findings were similar for spontaneous and oxytocininduced myometrial contractions. The conclusions are
therefore reliable and reproducible, indicating a potent
inhibitory effect of the peptide leptin. In addition, the
leptin levels investigated in this study include the normal
ranges of serum leptin measured in normal and obese
women.6,18 Upon conversion from ng/mL to molarity, it
is apparent that the normal levels of leptin in pregnancy
are in the nanomolar range, increasing by a factor of
more than 10 in obesity. This is fully covered by the
range of doses used in these experiments.
It is well established that maternal obesity is a major
mitigating factor against successful vaginal delivery,
after allowing for all other variables. We, and other
groups, have demonstrated that cesarean section rates
for women with a body mass index (BMI) indicating
obesity (ie O30), or morbid obesity (O35), are of the
order of 35% to 50%, which represents an odds ratio
(OR) of 2 to 3 in comparison to women of normal
BMI.1,2 Earlier reports have also outlined the significantly increased caesarean section rates associated with
obesity.19,20 Possible reasons for this have included increased maternal deposition of adipose tissue resulting
in physical obstruction to descent of the fetal presenting
part, fetal macrosomia, and inadequate pushing in the
second stage of labor. Our findings introduce the new
dimension that obesity can result in altered metabolic
modulation of human myometrial tissue, raising the
possibility that leptin, which is increased significantly
in association with obesity,6,21 contributes to inadequate
contractility of the uterus during labor. These results
provide a further explanation for the significant disparity that exists in successful vaginal delivery rates between women of normal BMI, and those who fall into
the obese categories.
The physiologic importance of leptin in pregnant
women is largely unknown. Leptin is increased in the
maternal blood particularly in the second trimester, with
a reduction in levels towards or at the time of parturition.13,22 Apart from its production in adipose tissue,
leptin is also produced in the placenta.8,10 Leptin is
known to have several mechanisms of functional activity
linked to autonomic, cardiovascular, inflammatory, and
endocrine pathways,4 aside from a possible direct effect
via intracellular signalling mechanisms. It is therefore difficult to speculate in relation to mechanism of action of
the uterorelaxant effect of leptin reported in this study.
Apart from future investigations relating to the
mechanism of leptin-induced myometrial inhibition,
there are other questions raised by this study. The
experiments reported here were all performed in lower
segment myometrium. While it is generally believed that
Moynihan et al
the functional aspects of lower segment myometrium
are similar to those of upper segment myometrium,23
this issue merits further investigation. The effects of leptin on nonpregnant myometrial contractility are also a
topic for future study. These findings also raise the possibility that other peptides and bioactive agents released
from the excessive adipose mass that occurs in obesity3
may modulate myometrial function.
Acknowledgments
We thank the Medical and Midwifery Staff at University
College Hospital Galway for their assistance with collection of the myometrium samples.
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