BIOLOGY OF REPRODUCTION 84, 167–178 (2011)
Published online before print 15 September 2010.
SIRT1 Is a Novel Regulator of Key Pathways of Human Labor1
Martha Lappas,2,3,4Amberlee Mitton,3,4Ratana Lim,3,4Gillian Barker,4,5Clyde Riley,3
and Michael Permezel3,4
Department of Obstetrics and Gynaecology,3University of Melbourne, Melbourne, Victoria, Australia
Mercy Perinatal Research Centre,4Mercy Hospital for Women, Heidelberg, Victoria, Australia
University of Queensland Centre for Clinical Research,5Brisbane, Queensland, Australia
Human sirtuin (SIRT) 1 and SIRT2, which possess nicotin-
amide adenosine dinucleotide (NAD+)-dependent deacetylase
activity, exhibit anti-inflammatory actions. However, there are
no data available on SIRT1 and SIRT2 expression and regulation
in human intrauterine tissues. Thus, the aim of this study was to
characterize the localization and expression of SIRT1 and SIRT2
in 1) placenta and fetal membranes before and after term
spontaneous labor onset, 2) prelabor fetal membranes from the
supracervical site (SCS) and a distal site (DS), and 3) in response
to proinflammatory stimuli. Further, the effect of SIRT activation
using resveratrol and SRT1720 on prolabor mediators was also
assessed. SIRT1 and SIRT2 were localized in the syncytiotro-
phoblast layer and the cytotrophoblasts of the placenta, amnion
epithelium, trophoblast layer of the chorion, and decidual cells.
Additionally, SIRT2 was found within the endothelial walls of
placental vessels. SIRT2, but not SIRT1, staining was significantly
lower in amnion and chorion obtained from the SCS compared
to a DS. On the other hand, SIRT1, but not SIRT2, gene and/or
protein expression was significantly lower in placenta, amnion,
and chorion obtained after labor compared to prelabor. SIRT1
expression, but not SIRT2, was down-regulated by lipopolysac-
charide (LPS) and proinflammatory cytokines TNF and IL1B. The
SIRT1 activators resveratrol and SRT1720 significantly de-
creased LPS-induced TNF, IL6, and IL8 gene expression and
release and PTGS2 mRNA expression and resultant prostaglan-
din (PG) E2and PGF2arelease from human gestational tissues. In
conclusion, SIRT1 possesses anti-inflammatory actions and thus
may play a role in regulating pregnancy and parturition.
cytokines, parturition, placenta, prostaglandins, SIRT1
Mammals possess seven sirtuins (SIRT1–7), which differ in
their subcellular localization, tissue distribution, and protein
substrates . They are classed according to their sequence of
amino acids: class 1 (SIRT1–3), class II (SIRT4), class III
(SIRT5) and class IV (SIRT6 and 7). SIRTs possess either
NADþ-dependent histone deacetylase (SIRT1, 2, 3, and 5) or
mono-ribosyltransferase (SIRT4 and 6) activity that contribute
to cellular regulation (reaction to stressors, longevity). Where
acetylation of core histones by histone acetyltransferases leads
to unwinding of DNA, to subsequently allow transcription
factors and RNA polymerase II to switch on gene transcription,
deacetylation of core histones is generally associated with
transcriptional repression [2, 3]. As such, SIRT1 and SIRT2,
have been implicated as a key mediator in the resolution of
inflammation . They have also been implicated in aging, cell
death/survival, metabolism, stress resistance and endocrine
signaling . Thus they may also play a role in human
parturition, however, to date; there is little data available about
the expression and regulation of SIRT proteins in human
gestational tissues. However, the one study demonstrates an
increase in SIRT1 in isolated amniotic epithelial cells in
response to stretch .
A successful outcome to labor and delivery at term is
dependent on three processes: 1) fetal maturation consistent with
extrauterine survival, 2) gestational and reproductive tract tissue
remodeling (either by proteases that degrade extracellular matrix
[ECM] or by apoptosis), and 3) the formation of uterotonic
agents that promote synchronous myometrial contractions [6–9].
Recent evidence from our own laboratory and others supports
the involvement of upstream coordinating regulators capable of
affecting the activity of the multiple terminal pathways involved
in spontaneous onset labor and delivery [10–14]. For example,
the nuclear transcription factors nuclear factor-jB (NFKB) and
the nuclear hormone receptor peroxisome proliferator activator
receptor (PPAR) appear to act in counterpoise to regulate the
expression of the terminal effector pathways involved in labor
and delivery: NFKB inducing expression while PPAR repress-
ing expression. Recent evidence has shown that SIRT proteins
negatively regulate the transcriptional activity of NFKB [15–
20], whereas they positively regulate the transcriptional activity
of PPAR . Additionally, SIRT1 is a negative regulator of
proinflammatory cytokines [18, 20] and matrix metalloprotei-
nase (MMP) 9 expression , which are required for processes
of human labor . As such, SIRTs are considered to have an
anti-inflammatory function. As the processes of human labor
exhibit all the hallmarks of an inflammatory response, SIRT
proteins may play a role in this process. However, our
knowledge concerning the expression, regulation, and functions
of SIRT proteins in vivo during late pregnancy and at the time of
labor is limited.
Thus, in this study, we use immunohistochemistry, Western
blotting, and quantitative real-time PCR to determine the
cellular distribution and gene and protein expression of SIRT1
and SIRT2 in human placenta, amnion, and choriodecidua
from normal-term pregnancies. In order to ascertain whether
SIRT proteins may play a role in the processes of human labor
and delivery, the expression of SIRT1 and SIRT2 is also
1Supported by the Melbourne Research Grant Scheme and ANZ
Charitable Trust (Medical Research and Technology Grant). Funding
for the Leica Qwin Image Analysis System was provided by the Medical
Research Foundation for Women and Babies. M.L. is a recipient of a
National Health and Medical Research Council (NHMRC) RD Wright
Fellowship (grant 454777).
2Correspondence: Martha Lappas, Department of Obstetrics and
Gynaecology, University of Melbourne, Mercy Hospital for Women,
Level 4/163 Studley Rd., Heidelberg, 3084, VIC Australia.
FAX: 61 3 8458 4380; e-mail: email@example.com
Received: 29 June 2010.
First decision: 5 August 2010.
Accepted: 7 September 2010.
? 2011 by the Society for the Study of Reproduction, Inc.
eISSN: 1529-7268 http://www.biolreprod.org
Downloaded from www.biolreprod.org.
studied in placenta and fetal membranes before and after term
spontaneous labor onset. Furthermore, we utilize the technique
we have established for the identification of fetal membranes
overlying the cervix (i.e., supracervical site [SCS]) in prelabor,
postdelivery tissues [22–24] to characterize the localization and
expression of SIRT1 and SIRT2 proteins in SCS fetal
membranes and compare them to distal fetal membranes in
order to better understand the mechanisms that may be
involved in rupture of fetal membranes. The regulation of
proinflammatory cytokines and prostaglandins are investigated
using two SIRT activators (SRT1720 and resveratrol).
MATERIALS AND METHODS
The Research Ethics Committee of Mercy Health approved this study.
Written, informed consent was obtained from participating women. The clinical
summary of patients used in this study is summarized in Table 1. There was no
difference in maternal age and body mass index, parity, or gestational age of the
patients recruited. Human placentae and attached fetal membranes were
obtained from women who delivered healthy, singleton infants at term (?37 wk
gestation) 1) before labor undergoing elective Cesarean section (indications for
Cesarean section were breech presentation and/or previous Cesarean section)
and 2) after spontaneous labor and normal vaginal delivery (n¼6–8 per group).
For the prelabor samples, identification of the SCS was performed as we have
previously detailed [22–24]. Briefly, prior to commencement of the Cesarean
section, a swab soaked in Bonneys Blue dye (1:1 mixture of brilliant green
[0.5% wt/vol] and crystal violet [0.5% wt/vol] dissolved in 90% ethanol vol/
vol) is introduced through the cervix onto the chorion lying above the internal
os of the cervix. On delivery of the placenta, a blue mark is obvious on the
membrane where the dye had been applied. Fetal membranes were collected
from both the SCS and from a distal site (DS) with no Bonneys Blue staining
(about 2 cm away from the periplacental edge). Postlabor fetal membranes were
identified and sampled in accordance with techniques originally described by
Malak and Bell ; amnion and underlying choriodecidua were obtained from
along the line of fetal membrane rupture. The placenta and fetal membranes
were collected within 30 min of application of Bonneys Blue and transferred
within 10 min of delivery to the laboratory in the hospital. A placental lobule
was removed from the central region of the placenta. The basal plate and
chorionic surface were removed from the placental tissue, and villous tissue
was obtained from the middle cross section. Placental tissue was blunt dissected
to remove visible connective tissue and calcium deposits. Tissue samples were
1) fixed and paraffin embedded for immunohistochemical analysis, 2) snap
frozen in liquid nitrogen and stored at ?808C for RNA and protein extraction,
or 3) used immediately for explant and cell culture experiments.
Tissue Explant Culture
Placental tissue (prepared as detailed previously) was placed in DMEM at
378C in a humidified atmosphere of 8% O2and 5% CO2for 1 h. Tissues were
blotted dry on sterile filter paper and transferred to 24-well tissue culture plates
(100 mg wet weight/well). The explants were incubated, in duplicate, in 2 ml
DMEM containing penicillin G (100 U/ml), streptomycin (100 lg/ml), and L-
glutamine (2 mM). To determine the effect of proinflammatory mediators on
SIRT expression, tissues were incubated in the presence of 10 lg/ml LPS, 20
ng/ml TNF, and 10 ng/ml IL1B. The chosen concentrations of LPS, TNF, and
IL1B are based on previously published studies . To determine the effect of
SIRT activation on proinflammatory cytokines, tissues were incubation, in the
absence or presence of 10 lg/ml LPS, with and without 1) 50, 100, and 200 lM
resveratrol and 2) 10 and 20 lM SRT1720. After 6 and/or 24 h of incubation,
tissues were collected and stored at ?808C until assayed for 1) expression of
nuclear RELA (NFKB p65) and cytoplasmic NFKBIA (IjB-a) proteins by
Western blotting, 2) nuclear RELA activity by ELISA, and 3) mRNA
expression by quantitative real-time PCR as detailed here. Media were collected
and stored at ?808C until assayed cytokine and prostaglandin release by
ELISA. Data were corrected for total protein and expressed as mean fold
change compared to LPS.
Gene Silencing of SIRT1 with siRNA in Primary
Primary amnion epithelial cultures were used to investigate the effect of
resveratrol on siRNA-mediated gene silencing of SIRT1 on proinflammatory
cytokine and prostaglandin gene expression. Cells were prepared as previously
described  and incubated in DMEM/F12 enriched with 20% FCS, 10 ng/ml
EGF, 2 mM L-glutamine, 100 U/ml penicillin G, and 100 lg/ml streptomycin.
Primary amnion cells (passage 1) at 40%–50% confluence in six-well plates
were transfected using TransIT-siQUEST reagent according to manufacturer
guidelines (Mirus Bio, Madison, WI). Mock transfected cells (transfection
reagent only) were used as the vehicle. Cells were transfected with 75 nM
SIRT1 siRNA (Ambion, Austin, TX) in DMEM/F12 with 5% FCS for 48 h.
The medium was then replaced with DMEM/F12 (with 1% FCS) containing 10
lg/ml LPS and 50 lM resveratrol, and the cells were incubated at 378C for 24
h. Cells and media were collected separately and stored at ?808C.
Fetal membrane tissues were rolled up (with amnion innermost), and all
rolls were placed in embedding cassettes (Techno-Plas, South Australia,
Australia), fixed in buffered formaldehyde solution (4% vol/vol), and
embedded in paraffin. Serial sections (4 lm thick) were cut and mounted on
sections onto superfrost plus slides. Sections were deparaffinized followed by
an antigen retrieval step (boiled in 10 mM Tris and 1 mM EDTA, pH 9.0, for
10 min followed by 20 min of incubation). Endogenous peroxidase activity was
removed using 3% H2O2(vol/vol) in methanol for 10 min. Sections were
transferred to tris-buffered saline (TBS; 20 mM Tris pH 7.6, 150 mM NaCl).
The sections were incubated in a humidity chamber for 1 h in antibody diluted
in 1% bovine serum albumin (BSA) in TBS (wt/vol). The primary antibodies,
purchased from Sigma (St. Louis, MO, USA), were rabbit polyclonal anti-
SIRT1 (S5322) and rabbit polyclonal anti-SIRT2 (S8447). Both SIRT1 and
SIRT2 were used at 10 lg/ml in 1% BSA/TBS (wt/vol). After incubation, the
binding sites were labeled with Dako Envisionþpolymer linked secondary
reagent and visualized using Dako DABþ(Dakocytomation, Carpinteria, CA).
Nuclei were counterstained with Mayers hematoxylin, and the sections were
dehydrated and coverslipped using a resinous mounting agent. Positive
controls, which were composite slides with tonsil, breast tumor, and ovarian
tumor, were included in each run. Negative control slides, where primary
antibody was replaced with normal rabbit IgG serum, were also included.
Sections were assessed microscopically for both intensity and extent of
staining. The entire tissue section was scored, and the extent of staining was
determined on a scale of 0–5 according to the estimated percentage of cells
stained: 0 , 10%, 1 ¼ 11%–25%, 2 ¼ 26%–50%, 3 ¼ 51%–75%, 4 ¼ 76%–
90%, and 5 . 90%. Staining intensity was assessed on a scale of 0–3: 0 ¼ no
staining, negative; 1 ¼ pale brown, weak; 2 ¼ brown, moderate; and 3 ¼ dark
brown, strong .
Assessment of SIRT1 and SIRT2 protein expression was analyzed by
Western blotting. The primary antibodies, purchased from Sigma, were rabbit
polyclonal anti-SIRT1 (S5322) and rabbit polyclonal anti-SIRT2 (S8447
cytoplasmic and nuclear protein was extracted as we have previously described
[29–31]). Assessment of NFKBIA protein expression was analyzed by Western
blotting. Rabbit polyclonal anti-RELA (sc-372), rabbit polyclonal anti-
NFKBIA, and horseradish peroxidase-conjugated goat anti-rabbit IgG were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Forty micrograms
of protein were separated on either 10.5%–14% or 7.5% criterion polyacryl-
amide gels (Bio-Rad Laboratories, Hercules, CA) and transferred to
polyvinylidine fluoride. Protein expression was identified by comparison with
the mobility of protein standard. Membranes were viewed and analyzed using
the Chemi-Doc system (Bio-Rad). Quantitative analysis of the relative density
of the bands in Western blots was performed using Quantity One 4.2.1 image
TABLE 1. Clinical characteristics of the patients.a
Clinical characteristicPrelabor (n ¼ 8)
32.8 6 1.9
23.6 6 0.8
38.7 6 0.2
3268 6 190
Postlabor (n ¼ 8)
27.8 6 3.2
21.8 6 0.8
39.3 6 0.3
3190 6 114
Maternal age (yr)
Maternal BMI (kg/m2)b
Gestational age at birth (wk)
Sex of infant (%)
4.2 6 1.0
2.3 6 0.6
3.0 6 0.8
2.2 6 0.4
aData present mean 6 SEM unless otherwise stated.
bBased on first antenatal visit at approximately 12 wk of gestation.
LAPPAS ET AL.
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Sirtuins have also been shown to regulate the transcriptional
activity of other transcription factors. SIRT1 deacetylates and
represses the activity of FOXO protein , whereas it
activates anti-inflammatory transcription factors, such as PPAR
. Previously, studies have demonstrated the importance of
these two transcription factors in the processes of human labor
and delivery [10–12, 14]; activation of PPARG leads to anti-
inflammatory actions in human gestational tissues [65–67].
Future studies determining the effect relationship between
SIRT1 and other transcriptions factors in human gestational
tissues are warranted.
Preterm labor and its consequences are the major contrib-
utors to perinatal morbidity and mortality worldwide. The
terminal pathways of spontaneous preterm and term labor are
the same: increased myometrial contractility and cervical
ripening, and fetal membrane rupture. A critical role for
cytokines, prostaglandins, and ECM remodeling enzymes in
these processes has been demonstrated. Term labor results from
physiologic activation of this pathway; preterm labor occurs
after pathologic activation, with intrauterine infection and/or
inflammation the most firmly established trigger. To our
knowledge, this is the first study to demonstrate a role for
SIRT1 as a key regulator of human parturition. Specifically,
human term spontaneous labor is associated with decreased
SIRT1 levels in human gestational tissues. We also show that
SIRT1 is anti-inflammatory; it is down-regulated by prolabor
cytokines and bacterial endotoxin, and prolabor mediators are
down-regulated by SIRT1 activation and up-regulated by
SIRT1 inhibition. Collectively, these data suggest that SIRT1
may be involved in the maintenance of pregnancy and
parturition and, as such, presents an attractive therapeutic
target for preterm labor. Indeed, previous studies have shown
SIRT1 to be essential for normal embryogenesis; Sirt1
knockout mice have a severe phenotype, with a high degree
of embryonic and postnatal lethality [68, 69]. Future studies
defining the signaling pathways upstream and downstream of
SIRT1 and characterizing their roles in human pregnancy are
warranted. An understanding of SIRT1 and its biology in the
processes of human labor will provide new opportunities for
developing more effective therapeutic approaches to treat
William E. Ackerman IV and Douglas A. Kniss (Department of
Obstetrics and Gynecology, College of Medicine, Ohio State University,
Columbus, OH, USA) are thanked for their input into the resveratrol
studies. We would like to thank Mahalia Chai and Dr. Nicole Reti (from
the Department of Obstetrics and Gynaecology, University of Melbourne,
Mercy Hospital for Women) for their assistance with the fetal membrane
sample collection. We would also like to thank Nicole Dellios (University
of Queensland) for providing the primary amnion cells. The authors
gratefully acknowledge the assistance of the Clinical Research Midwives
Gabrielle Fleming, Astrid Tiefholz, and Anne Beeston and the obstetrics
and midwifery staff of the Mercy Hospital for Women for their
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