NEW RESEARCH HORIZON Review

Epigenetic regulation of endometrium

during the menstrual cycle
S.K. Munro
1,2
, C.M. Farquhar
3
, M.D. Mitchell
1,2,4
,
and A.P. Ponnampalam
1,*
1
The Liggins Institute, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
2
The National Research Centre for Growth
and Development, c/- The Liggins Institute, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
3
Department of
Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1142,
New Zealand
4
University of Queensland Centre for Clinical Research, RBWH Campus, Herston, Brisbane, QLD 4029, Australia
*Correspondence address. E-mail: a.ponnampalam@auckland.ac.nz
Submitted on November 3, 2009; resubmitted on December 22, 2009; accepted on February 2, 2010
abstract: The endometrium undergoes morphological and functional changes during the menstrual cycle which are essential for uterine
receptivity. These changes are driven by estrogen and progesterone and involve the ne control of many different genesseveral of which
have been identied as being epigenetically regulated. Epigenetic modication may therefore inuence the functional changes in the endo-
metrium required for successful implantation. There is, however, only limited information on epigenetic regulation in endometrium. We
review the potential role of epigenetic regulation of key processes during the menstrual cycle and present our own ndings following a pre-
liminary study into global acetylation levels in the human endometrium. A changing epigenetic state is associated with the differentiation of
stem cells into different lineages and thus may be involved in endometrial regeneration. Histone acetylation is implicated in the vascular endo-
thelial growth factor pathway during angiogenesis, and studies using histone deacetylase inhibitors suggest an involvement in endometrial
proliferation and differentiation. The processes of decidualization and implantation are also associated with epigenetic change and epigenetic
modulators show variable expression across the menstrual cycle. Our own studies found that endometrial global histone acetylation, as
determined by western blotting, changed throughout the menstrual cycle and correlated well with expected transcription activity during
the different phases. This suggests that epigenetics may be involved in the regulation of endometrial gene expression during the menstrual
cycle and that abnormal epigenetic modications may therefore be associated with implantation failure and early pregnancy loss as well as
with other endometrial pathologies.
Key words: endometrium / epigenetics / histone acetylation / menstrual cycle / uterus
Introduction
Implantation is a highly controlled process, involving a dialogue between
the endometrium and the implanting embryo which is crucial for the
establishment and maintenance of pregnancy (Norwitz et al., 2001;
Dey et al., 2004; Makker and Singh, 2006; Palis et al., 2007). Little is
known about the regulation of implantation (Makker and Singh,
2006), however inadequate uterine receptivity is thought to be respon-
sible for two-thirds of implantation failures (Simon et al., 1998). Control
of gene expression is crucial to the developmental processes which are
regularly implemented as the endometrium undergoes cyclic periods of
growth and differentiation. Inappropriate epigenetic modication result-
ing in aberrant expression of endometrial regulatory genes or proteins
may therefore provide a partial explanation for the failure of embryo
implantation (Kao et al., 2003).
Deregulation of implantation may have consequences beyond the
failure of implantation and subsequent infertility. In mice, even a transi-
ent postponement of blastocyst attachment is sufcient to cause a
detrimental ripple effect throughout the pregnancy, with aberrant
spacing of embryos, defective placentation, resorption and retarded
development of foetuses observed (Wilcox et al., 1999; Song et al.,
2002; Ye et al., 2005). Poor implantation and placentation have also
been associated with end results such as intrauterine growth restriction,
pre-eclampsia, and preterm birth in humans (Wang and Dey, 2006).
Epigenetic modication, resulting in altered chromatin structure and
transcriptional activity, controls gene expression and may therefore
control the functional changes in the endometrium which occur
throughout the menstrual cycle. Here, we examine the role epige-
netics plays in several key processes which occur during the menstrual
cycle and the establishment of pregnancy and discuss whether
normal human endometrium could be under epigenetic regulation.
Epigenetics
For mainly historical reasons, there has been some confusion about
what epigenetics actually refers to. A standard denition has been
mitotically and/or meiotically heritable changes in gene function
& The Author 2010. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
For Permissions, please email: journals.permissions@oxfordjournals.org
Molecular Human Reproduction, Vol.16, No.5 pp. 297310, 2010
Advanced Access publication on February 5, 2010 doi:10.1093/molehr/gaq010

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that cannot be explained by changes in gene sequence (Russo et al.,
1996). Of these, heritability is perhaps the most restrictive require-
ment: for example, although neurons undergo stable alterations,
since they rarely divide the changes are not heritable in the simplest
sense; whereas other alterations seem to be heritable, but are not
maintained in a stable manner. These issues led to a proposed de-
nition of epigenetics as the structural adaptation of chromosomal
regions so as to register, signal or perpetuate altered activity states
(Bird, 2007), and more recently; an epigenetic trait is a stably inher-
ited phenotype resulting from changes in a chromosome without
alterations in the DNA sequence (Berger et al., 2009).
Changes in chromatin architecture can be mediated by DNA
methylation and the post-translational modication of histone tails
(Dolinoy et al., 2007; Jirtle and Skinner, 2007). These histone modi-
cations are also involved in the recruitment of transcription factor
complexes (Berger, 2007) and therefore act to regulate gene
expression. It is thought that non-protein coding RNAs may also regu-
late chromatin structure, with RNA mediated interplay between the
environment, epigenome and transcriptome also occurring, however
this is still an emerging area of research (Mattick et al., 2009). Cellular
components such as DNA methyltransferases (DNMTs), histone dea-
cetylases (HDACs) and histone acetyltransferases (HATs) act to main-
tain or alter these modications as appropriate. Two of the most
well-known epigenetic-related phenomena are X chromosome inacti-
vation and genomic imprinting (Jirtle and Skinner, 2007). A range of
environmental factors, including nutrition and exposure to xenobiotic
chemicals, can inuence the establishment and maintenance of epige-
netic patterning and thus long-term gene transcription (Dolinoy et al.,
2007; Jirtle and Skinner, 2007).
Both DNA methylation and histone modications have been found
to be altered in human cancers within the promoter regions of tumour
suppressor genes and oncogenes (Jirtle and Skinner, 2007). It is
becoming increasingly clear that such changes are also associated
with other disease states (Guo, 2009; Trenkmann et al., 2009;
Turunen et al., 2009).
DNA methylation
DNA methylation is the covalent modication of post-replicative DNA
by the addition of a methyl group to the cytosine ring to form methyl
cytosine; a process which is catalyzed by DNMTs (Ohgane et al.,
2008). Three main DNMTs are present in mammals: DNMT1,
which maintains methylation by recognizing uni-strand methylation
after replication, and DNMTs 3a and 3b, which predominantly func-
tion as de novo methyltransferases acting at previously un-methylated
sites (Brero et al., 2006).
In mammals, DNA methylation is thought to be exclusively associ-
ated with cytosine-phosphate-guanine (CpG) dinucleotides, occurring
on both strands at the cytosine residue (Brero et al., 2006). In ver-
tebrates, large un-methylated GC rich regions can be found at the
5
0
end of many genes and are termed CpG islands (Bird, 1987).
DNA methylation at promoter associated CpG islands is strongly
linked to repression of transcriptional activity; however, the precise
role of CpG methylation is an area of ongoing research (Bird,
2002). Approximately 1020% of genes display DNA methylation
patterns in a tissue-specic manner (Song et al., 2005; Eckhardt
et al., 2006; Rakyan et al., 2008) and these so-called tissue-specic dif-
ferentially methylated regions have been associated with tissue-specic
patterns of gene expression (Rakyan et al., 2008). The repressive
nature of DNA methylation is thought to result from either the
methyl group inhibiting the binding of regulatory molecules to a
CpG site or by acting as a recognition signal for methyl-CpG-binding
proteins with repressive properties (Bird, 2002), both of which are
mechanisms likely to act in a site-specic manner. However, it
should be noted that DNA methylation is not always associated
with transcriptional repression. A recent study conrmed previous
ndings of a negative correlation between DNA methylation and
gene expression at promoters with a medium or high CpG density,
however, contrary to previous notions, even some low-CpG density
promoters showed this correlation. In contrast, gene-body methyl-
ation was found to positively correlate with gene expression suggesting
novel roles for DNA methylation (Rakyan et al., 2008). Further
research is required to elucidate the sequence and context-specic
nature of this epigenetic modication.
Histone modications
Histones are integral to the higher order structure of chromatin.
Approximately 146 bp of DNA is wrapped around a histone
octamer consisting of two sets of the core histonesH2A, H2B,
H3 and H4to form a nucleosome. These nucleosomes are linked
by loops of DNA and the linker histone H1. Although the globular
domains of histones associate closely with each other, the trailing
amino acid tails protrude past the surrounding DNA and are
subject to post-translational modication. New histone proteins are
produced during the S (synthesis) phase of the cell cycle when
DNA is being replicated (Lucchini and Sogo, 1995). The existing his-
tones from the parental strand are randomly segregated between par-
ental and daughter strands during replication and the rest of the
histones, synthesized de novo, are then deposited (Sogo et al.,
1986). Two H3H4 heterodimers are deposited either sequentially
or as a heterotetramer, followed by the two H2AH2B dimers,
thus forming the octamer (Worcel et al., 1978; English et al., 2006).
Many distinct modications of histones exist, with those identied
including lysine acetylation, lysine methylation, arginine methylation,
phosphorylation, ubiquitylation, sumoylation, ADP ribosylation, deimi-
nation and proline isomerization (Kouzarides, 2007; Lindner, 2008).
Modication of histones can be difcult to study as histone modi-
cations are very dynamic. All known histone post-translational modi-
cations have now been shown to be reversible, and as some histone
modications have been shown to change within minutes of a stimulus,
it is uncertain as to which modications can be considered as truly
stable and therefore epigenetic. It is also unknown how the persist-
ence of chromatin state is achieved, and which modications are
therefore truly heritable (Berger, 2007; Kouzarides, 2007).
Histone deacetylation, one of the best characterized histone modi-
cations, is associated with gene silencing, whereas histone acetylation
is associated with transcriptional activation (Fuks, 2005) (Fig. 1).
Histone acetylation patterns are maintained by two opposing groups
of enzymes, the HATs and HDACs (Hildmann et al., 2007). There
is also evidence for crosstalk between elements of the DNA methyl-
ation machinery and those involved in histone modication, with
DNMTs able to recruit complexes containing HDACs (Fuks, 2005).
A group of proteins with methyl binding domains are also able to
recognize methylated DNA sequences and recruit HDACs (Nan
et al., 1998; Irvine et al., 2002; Jorgensen and Bird, 2002). A reverse
298 Munro et al.

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regulatory pathway whereby histone acetylation mediates DNA
demethylation may also exist (Szyf et al., 1985; Selker, 1998;
Cervoni and Szyf, 2001) as some data show that HDAC inhibition
can lead to changes in DNA methylation (Cosgrove and Cox, 1990;
Chen and Pikaard, 1997; Hu et al., 1998; Selker, 1998; Hu et al.,
2000; Dobosy and Selker, 2001; Maass et al., 2002), although, this is
not always the case (Cameron et al., 1999; Singal et al., 2001;
El-Osta et al., 2002). HDACs and HATs, like many other enzymes
involved in histone modication, have actions which are not specic
to the histones and are able to modify other proteins. Over 60 tran-
scription factors including the tumour suppressor p53 are subjected to
acetylation and some HATs also physically associate with non-protein
coding RNA (Yang and Seto, 2007). Similar actions are seen with
other histone modifying enzymes, such as lysine-specic
demethylase-1, which in addition to histone demethylation, has
been found to be involved in the demethylation and stabilization of
DNMT1, which is required for the maintenance of global DNA
methylation (Hotz and Peters, 2009; Wang et al., 2009).
The menstrual cycle
Human endometrium undergoes cyclic morphological changes invol-
ving precise periods of growth, differentiation and regression, with
each new menstrual cycle starting again on average every 28 days
during a womans reproductive years (Curry and Osteen, 2003).
These changes are the visible result of the interactions of an array
of biological communication pathways and many local factors including
cytokines, which are under the overall control of the ovarian steroids
estrogen and progesterone (Curry and Osteen, 2003). Rising serum
estrogen levels from the ovary stimulate endometrial growth during
the proliferative phase. Epithelial and stromal cells from the functional
layer proliferate extensively, with both cell types increasingly acquiring
receptors to both estrogen and progesterone in parallel to the rise in
estrogen levels (Garcia et al., 1988). Following ovulation, usually occur-
ring around Day 14 of the menstrual cycle, there is a secretory trans-
formation of the endometrium in response to rising serum
progesterone levels produced by the corpus luteum (Brenner and
West, 1975; Lessey, 2000). Changes in the glandular epithelium are
apparent by cycle Days 1516 (Noyes et al., 1950; Lessey, 2000). Fol-
lowing fertilization, signals from the developing blastocyst act in
concert with steroid hormones to induce a receptive state supportive
of implantation (Paria et al., 2002; Makker and Singh, 2006). The endo-
metrial epithelium only transiently acquires this receptive state and a
variety of architectural, cellular, molecular and biochemical events
must be coordinated (Makker and Singh, 2006). The endometrial
stroma becomes more vascular and oedematous during this phase,
with the glands displaying enhanced secretory activity (Norwitz
et al., 2001). Additional morphological changes occur with the
process of decidualization by which stromal cells differentiate to
support pregnancy (Carson et al., 2000). The secretory phase will,
in the absence of an embryo, terminate with the shedding of the func-
tionalis layer approximately 2 weeks after ovulation, a process known
as menstruation (Carson et al., 2000). As menstruation results in
bleeding and tissue loss, it is followed by a period of repair and regen-
eration of the functionalis prior to the next cycle of proliferation. The
regenerative capacity of the endometrium is such that within a cycle it
grows to a thickness of 57 mm from the initial 0.51 mm post-
menstruation (McLennan and Rydell, 1965). Consequently, the
endometrium requires a continuing activation of processes such as
angiogenesis that are normally only associated with early development
(Curry and Osteen, 2003). As many of these processes have also been
Figure 1 Histone post-translational modications (PTMs) inuence chromatin structure. This leads to either a more condensed state, associated
with transcriptional repression or a more relaxed state associated with transcriptional activation. PTMs are the net result from activity of epigenetic
modulators which form large complexes with some adding, others removing different modications. Chromatin state is also linked to the degree of
DNA methylation with crosstalk between elements of the DNA methylation machinery and those involved in histone modication.
Epigenetic regulation of human endometrium 299

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associated with epigenetic alterations, we propose that the endome-
trium may be subject to epigenetic regulation throughout the men-
strual cycle (Fig. 2).
Regeneration of the endometrium
The stratum basalis or basal layer of the endometrium remains intact
throughout the menstrual cycle and contains the cells from which the
functional layer will regenerate following menstruation (Noyes et al.,
1950; Curry and Osteen, 2003; Diedrich et al., 2007). The regenera-
tive capacity of human endometrium led to the proposal that there
might be a local population of adult stem cells contributing to the
regeneration of the functional layer (Padykula, 1991) (Fig. 2). Adult
stem cells are small populations of quiescent cells that undergo asym-
metrical cell divisions when activated, allowing the maintenance of
their own population in addition to the production of daughter
cellslike embryonic stem cellsare likely to be under the control
of DNA methylation and chromatin modications. These adult stem
cells/progenitor cells can proliferate extensively (Fuchs and Segre,
2000) differentiating to form the different cell types of that lineage
and represent a more differentiated version of the stem cells seen
in early development.
It is clear that there are major epigenetic alterations of the genome
during mammalian development and during differentiation of embryo-
nic stem cells (Reik, 2007). Embryonic stem cells differ in their global
gene expression prole from the more lineage restricted adult stem
cells, with a gradual loss of pluripotent gene expression and gain of
lineage-specic genes occurring during differentiation (Perry et al.,
2004; Yeo et al., 2007; Atkinson and Armstrong, 2008; Zardo et al.,
2008). Changes in chromatin architecture are thought to be respon-
sible for these altered gene expression proles and thus stem cell iden-
tity, with the epigenetic code determining a stem cells
responsiveness to developmental signals (Cirillo et al., 2002; Schrem
et al., 2002; Zaret et al., 2008; Collas, 2009). External stimuli such
as extracellular growth factors may also result in direct alteration of
the chromatin status, impeding or facilitating stem cell differentiation
(Song and Ghosh, 2004; Snykers et al., 2009). The unique properties
of stem/progenitor cells, pluripotent differentiation and self-renewal
seem to be under the control of DNA methylation and chromatin
modications (Shukla et al., 2008). In light of this, it is not surprising
that alteration of epigenetic state has been found to play an important
role in the reprogramming of cell fate, with studies showing that vir-
tually any cell can be regressed to a less mature state with the right
combination of factors (Cowan et al., 2005; Ivanova et al., 2006;
Takahashi et al., 2007; Mikkelsen et al., 2008; Zardo et al., 2008).
Of note is that most human cancers are associated with altered
epigenetic patterns in tissue stem/progenitor cells (Feinberg et al.,
2006) emphasizing the importance of epigenetics in control of cell
proliferation and differentiation.
The rst endometrial stem/progenitor cell candidates were ident-
ied in 2004, with a small population of both human endometrial epi-
thelial cells and stromal cells shown to possess clonogenic activity
in vitro (Chan et al., 2004). It has been suggested that these colony-
forming unit (CFU) cells might be responsible for the regeneration
of both cycling and atrophic endometrium (Gargett et al., 2008),
with CFU cells being found in normal cycling endometrium, inactive
peri-menopausal endometrium and in the endometrium of women
taking oral contraceptives (Schwab et al., 2005). The existence of epi-
thelial and stromal stem/progenitor cells in human and mouse endo-
metrium have been conrmed using stem cell activity assays (Gargett
et al., 2007; Gargett et al., 2009). Assays also recently identied differ-
ent CFU cells as being of an epithelial progenitor cell type (Gargett
et al., 2009), which is expected to reside somewhere in the basalis
layer (Gargett, 2007), and an endometrial mesenchymal stem/
progenitor cell type (Reynolds and Rietze, 2005; Dimitrov et al.,
2008; Gargett et al., 2009) recently localized to perivascular niches
in both the basal and functional layers of the endometrium (Schwab
and Gargett, 2007). Epithelial side population cells have also
been identied in human endometrial cell suspensions and cultures
Figure 2 Many individual events during the menstrual cycle are associated with epigenetic change. Epigenetics has been implicated in the processes
of stem cell differentiation, angiogenesis, implantation and decidualization, and it is likely that given the dynamic nature of the endometrium, that it is
under epigenetic regulation.
300 Munro et al.

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(Kato et al., 2007; Tsuji et al., 2008) and are reported to be capable of
reconstituting various endometrial components and structures when
xenotransplanted into mice (Maruyama, 2009).
It has been proposed that inappropriate shedding of endometrial
progenitor cells may contribute to the development of endometriotic
lesions, with evidence of monoclonality (Jimbo et al., 1997; Starzinski-
Powitz et al., 2001; Leyendecker et al., 2002; Tanaka et al., 2003; Wu
et al., 2003; Gargett, 2007; Gargett et al., 2008). With a change in
environment following retrograde menstruation potentially altering
both paracrine and endocrine signalling, it is likely that a number of
epigenetic changes arise in ectopic endometrium, consequently
giving rise to the pathology of endometriosis. Indeed, endometriosis
is increasingly being identied as an epigenetic disease and the evi-
dence for this has recently been reviewed (Guo, 2009). Evidence of
distinct epigenetic changes arising in endometriosis suggests a mechan-
ism by which key endometrial genes involved in proliferation and
differentiation may already be under epigenetic control, with acti-
vation/repression in response to environmental cues. It is likely that
epigenetic changes assist in the regulation of progenitor cell differen-
tiation into the variety of lineage-restricted endometrial cell types
required during regeneration. Interestingly, the initial stages of endo-
metrial repair do not appear to require estrogen. Within 48 h of shed-
ding, when circulating estrogen levels are low, epithelial cells migrate
over the denuded surface of the endometrium (Okulicz and Scarrell,
1998). At this time, endometrial epithelial cells also lack the
expression of estrogen receptor alpha (ERa). A mouse model of
endometrial restoration following menstruation has conrmed regen-
eration can occur in the absence of estrogen (Kaituu-Lino et al.,
2007). Rapid proliferation of epithelial label-retaining cells in response
to estrogen despite the lack of receptor indicates a transmission of
proliferative signals from stromal niche cells (which do express ERa)
to candidate stem/progenitor cells (Chan and Gargett, 2006;
Gargett, 2007) and exemplies the importance of local environment
in the control of endometrial repair and regeneration.
Proliferation
During the proliferative phase, rising serum estrogen levels from the
ovary stimulate endometrial growth with extensive proliferation of
the epithelial and stromal cells of the functional layer, which increas-
ingly acquire receptors to both estrogen and progesterone (Garcia
et al., 1988). Studies into endometrial cancer have allowed us some
insight into the potential for epigenetic regulation of normal endo-
metrial proliferation. For example promoter hypo-methylation of the
transcription factor paired-box gene 2 (PAX2) has been identied in
endometrial cancer, with 75% of endometrial carcinoma in one
study exhibiting PAX2 promoter hypo-methylation (Wu et al.,
2005a). This hypo-methylation was found to allow inappropriate
activation by estrogen and tamoxifen, and resulted in enhanced cell
proliferation (Wu et al., 2005a). This suggests that endometrial
responsiveness to estrogen signalling may be dependent not only on
receptor expression but also on the appropriate epigenetic modi-
cation of other effectors. Receptiveness to steroid hormones and
their antagonists is a particular problem with endometrial tissue, par-
ticularly in patients receiving treatment for breast cancer. Tamoxifen, a
selective ER modulator which acts as an ER antagonist in the breast
and is used for breast cancer therapy, acts as a partial agonist in the
endometrium (Gallo and Kaufman, 1997) leading to an increased
risk of developing uterine adenocarcinoma (Fisher et al., 1994). Treat-
ment with the HDAC; Valproic acid (VPA) has been shown to block
the proliferation of uterine endometrial cells exposed to both tamox-
ifen and estrogen in Ishikawa cells (Hodges-Gallagher et al., 2007). The
Ishikawa cell line is known to be heterogeneous (Nishida et al., 1996),
which may account for reports of VPA both stimulating (Graziani et al.,
2003) and inhibiting (Takai et al., 2004) proliferation in Ishikawa cells,
but this does indicate a potential link between endometrial prolifera-
tive capacity and histone acetylation status.
Angiogenesis
Angiogenesis is the process of new blood vessel growth and occurs reg-
ularly during every menstrual cycle in human endometrium, with the
growth and regression of blood vessels occurring under the inuence
of estrogen and progesterone. The regulation of angiogenesis in the
endometrium has been difcult to elucidate, with a large number of
angiogenic factors being secreted. The direct correlation of these
factors with endometrial angiogenesis is hard to conrm as endothelial
cell proliferation, a marker for angiogenic activity, occurs throughout
the menstrual cycle (Gargett and Rogers, 2001; Rogers et al., 2009). In
animal models, endothelial cell proliferation is closely linked to circulating
levels of estrogen and progesterone; however, regulation is not so simple
in humans, with evidence that estrogen can both promote and inhibit
angiogenesis under different circumstances (Girling and Rogers, 2005).
In mouse models, endothelial cell proliferation occurs following the
elevation of estrogen levels post-estrus, and in conjunction with rising
plasma progesterone levels (Walter et al., 2005). In ovariectomized
mice, this proliferation can be induced to occur within 24 hours by
injection with 100 ng of estrogen 7 days post-surgery (Heryanto and
Rogers, 2002). Treatment with either antibodies against vascular
endothelial growth factor (VEGF) or VEGF receptor 2 inhibitors, com-
pletely blocks this response (Heryanto et al., 2003), suggesting that
estrogen acts through the VEGF pathway. An inux of VEGF expres-
sing neutrophils into the endometrium also occurs following estrogen
injection, which when blocked, results in decreased proliferation of
endothelial cells in response to estrogen implicating circulating leuco-
cytes in the mediation of this response (Heryanto et al., 2004). Pro-
gesterone injection alone, without estrogen priming, also stimulated
endothelial cell proliferation, although this effect is only mediated in
part through the VEGF pathway (Walter et al., 2005).
In the human endometrium, VEGF is the principle angiogenic factor
and its action is not restricted to vascular smooth muscle cells
(Charnock-Jones et al., 1993). VEGF mRNA is expressed mainly in
the stroma during the proliferative phase (Li et al., 1994; Kawano
et al., 2000), with a subset of stromal cells exhibiting much higher
expression, and a low level of expression is also observed in the gland-
ular epithelium (Charnock-Jones et al., 1993). During the secretory
phase stromal VEGF expression is more uniform and glandular
expression is increased (Charnock-Jones et al., 1993), with overall
expression three to ve times higher in the secretory phase when
compared with the early proliferative phase (Charnock-Jones et al.,
1994). A high level of VEGF mRNA can also be detected in menstrual
tissue (Charnock-Jones et al., 1993). Focal VEGF is also located in neu-
trophils associated with micro-vessel walls (Gargett et al., 2001). In
stromal cells, VEGF production is accompanied by the production of
matrix metalloproteinases (MMPs) which are involved in extracellular
matrix degradation and regulation of angiogenesis (Li et al., 1994;
Epigenetic regulation of human endometrium 301

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Moses and Langer, 1991; Kawano et al., 2000). Treatment with
thrombin leads to increased VEGF, MMP-1 and active MMP-2, and
this up-regulation appears to act through PAR-1 (a member of the
G-protein-coupled protease-activated receptors) and mitogen-
activated protein kinase (Furukawa et al., 2009).
Evidence fromother tissues suggests that epigenetic mechanisms play
a role in the process of angiogenesis. For example, studies have shown
that VEGF actions on endothelial cell proliferation are mediated through
regulation of the HDAC-7. Like the other mammalian Class II HDACs,
HDAC-7 is responsive to extracellular signals and contains an N-
terminal extension that interacts with other transcription cofactors
(Verdin et al., 2003; Wang et al., 2008). Phosphorylation of conserved
serine residues in the N-terminal domain result in the removal of this
class of HDACs from the nucleus to the cytoplasm, activating gene
expression (Grozinger and Schreiber, 2000; McKinsey et al., 2000;
Vega et al., 2004; Mottet et al., 2007; Ha et al., 2008; Wang et al.,
2008), and blockade of HDAC-7 phosphorylation represses VEGF-
mediated endothelial cell proliferation and migration (Wang et al.,
2008). HDAC-7 can maintain vascular integrity by repressing MMP-10
expression in endothelial cells and in vitro assays support a role for
the modulation of angiogenesis by HDAC-7 (Chang et al., 2006;
Mottet et al., 2007). Inhibition of HDAC-7 gene transcription results
in altered endothelial cell morphology, migration and capillary forming
capacity but does not affect proliferation, adhesion or apoptosis
(Mottet et al., 2007). It also resulted in the up-regulation of platelet-
derived growth factor-B (PDGF-B) and its receptor. Inhibition of
HDACs blocks the differentiation of endothelial progenitor cells and
this appears to act through the down-regulation of homeobox
protein HOXA9, as over expression of HOXA9 partially rescues the
differentiation blockade observed after HDAC inhibitor treatment
(Rossig et al., 2005). HOXA9 appears to act as a regulator of
endothelial-committed genes such as endothelial nitric oxide synthase,
VEGFR-2 and VE-cadherin, and also mediates the shear stress-induced
maturation of endothelial cells (Rossig et al., 2005). This implicates
histone acetylation in the control of angiogenesis, with VEGF signalling
mediated through HDAC-7 resulting in the up-regulation of a number
of genes critical to angiogenesis, and it is possible that similar mechan-
isms may be acting in the endometrium (Fig. 2). HOXA9 mRNA is
expressed in human endometrium with a decrease in expression
reported between the early and mid-secretory phase (Carson et al.,
2002). Interestingly, the HOXA9 gene in endometrium has been
shown to be methylated in women with ovarian cancer, which is
thought to result from abnormal methylation of HOXA9 in mullerian
tract stem cells (Widschwendter et al., 2009). PDGF-B transcripts
have been identied in human endometrium (Boehm et al., 1990),
and both PGDF-B and MMP10 are highly expressed by a population
of cells isolated frommenstrual blood termed endometrial regenerative
cells (ERC) which were capable of differentiation into a number of
lineages including epithelial and endothelial lineages (Meng et al.,
2007). In addition, MMP10 production by endometrial stromal cells
has been shown to increase in response to conditioned media from
human trophoblasts (Hess et al., 2007), which may assist in vascular
remodelling associated with placentation.
Differentiation
Rising serum progesterone levels post-ovulation result in a secretory
transformation of the newly regenerated endometrium as the different
cell types begin to differentiate (Brenner and West, 1975; Lessey,
2000). Studies involving Ishikawa cells suggest a role for histone acety-
lation in the control of endometrial differentiation. Although Ishikawa
cells are a well-differentiated adenocarcinoma cell line rather than a
normal endometrial cell line type (Nishida, 2002), treatment with
the related HDAC inhibitors Trichostatin A (TSA) and suberoylanilide
hydroxamic acid (SAHA), results in differentiation which closely
resemble normal secretory phase endometrial epithelium in a time-
and dose-dependent manner. This is accompanied by the expression
of secretory phase-specic proteins (Uchida et al., 2005b) and the
effects on proliferation and differentiation by TSA and SAHA were
comparable to treatment with estrogen and progesterone suggesting
a parallel between histone hyper-acetylation, differentiation and
secretory phase hormone exposure. Surprisingly, the HDAC inhibitor
effects could be blocked by gene silencing of glycodelin, an established
differentiation marker for endometrial glandular cells (Seppala et al.,
2002) which suggests some level of feedback.
Decidualization is a process of differentiation of the broblast-like
stromal cells of the endometrium and is necessary to support the
developing embryo (Sakai et al., 2003). These cells, primed by estro-
gen in the proliferative stage, respond to progesterone by becoming
larger and rounder and initiating a range of biochemical pathways.
Decidual cells arise rst in the vicinity of the spiral arteries, and
begin to spread throughout the endometrium during the secretory
phase of the menstrual cycle. Following implantation of an embryo,
decidualization extends throughout the stroma, resulting in the for-
mation of the decidua characteristic of pregnancy (Noyes et al.,
1950; Sakai et al., 2003).
The process of decidualization has been associated with epigenetic
changes in vitro (Fig. 2). HDAC inhibitor treatment of human endo-
metrial stromal cells with TSA resulted in morphological change and
expression of differentiation marker proteins such as insulin-like
growth factor (IGFBP-1) and prolactin, indicative of decidualization
(Sakai et al., 2003). It was found that histones H3 and H4 became
acetylated upon decidualization and acetylated H4 in turn was associ-
ated with the ovarian steroid induced promoter activation of IGFBP-1
(Sakai et al., 2003). In addition, human endometrial adenocarcinoma
cell lines treated with TSA or SAHA have been shown to undergo
morphological transformation to a attened and widespread epithelial
phenotype, with induction of differentiation markers such as glycode-
lin, leukaemia inhibitory factor (LIF), interleukin-1 receptor and glyco-
gen synthesissimilar to responses seen with ovarian steroid
hormones (Uchida et al., 2005a).
Implantation
With epigenetics implicated in the control of decidualization (Sakai
et al., 2003; Uchida et al., 2007b), it is likely that epigenetic regulation
plays a role in the establishment of endometrial receptivity and in sub-
sequent embryo implantation (Fig. 2). DNA methylation and histone
acetylation have been directly correlated with the expression of
implantation related genes in other contexts, including LIF (Uchida
et al., 2005b), HOXA10 (Wu et al., 2005b; Yoshida et al., 2006), gly-
codelin (Uchida et al., 2005b; Uchida et al., 2007a), MMPs, tissue
inhibitors of matrix metalloproteinase (Clark et al., 2007), E-cadherin
(Yoshiura et al., 1995; Saito et al., 2003; Rahnama et al., 2006;
Rahnama et al., 2009) and mucin (MUC)1 (Yamada et al., 2008).
The steroid hormone receptors themselves are susceptible to
302 Munro et al.

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epigenetic modulation with promoter associated CpG islands
(Campan et al., 2006) and are capable of inducing the modication
to the chromatin structure of other genes (Leu et al., 2004). In turn
these epigenetically modied genes are involved in the expression of
MUC1 (Horne et al., 2006), the b3 subunit (Achache and Revel,
2006), Osteopontin (Makker and Singh, 2006), heparin-binding epi-
dermal growth factor (Makker and Singh, 2006) and MMPs and has
been linked to the formation of pinopodes (Bagot et al., 2001;
Aghajanova et al., 2003), with many secondary interactions between
these factors. It is conceivable that many other genes involved are
similarly regulated.
Expression of epigenetic modulators during
the menstrual cycle
Signicant changes in the expressions of DNMTs have been observed
in endometrium during the menstrual cycle with a reported decrease
in DNMT mRNA during mid-late secretory phase, and following
estrogen and medroxy-progesterone acetate treatment in stromal
cell cultures (Yamagata et al., 2009). DNMT protein levels in human
endometrium remain less clear, with reports of no change in
DNMT expression during the menstrual cycle by one group (Yamagata
et al., 2009) and that DNMT1 expression is restricted to the prolifera-
tive phase by another (Liao et al., 2008). In proliferative endometrium,
immunostaining reveals a high level of methylated cytosine residues in
cells of the glandular epithelium and the stroma. In the secretory phase
levels of methylated cytosines in the cells of the glandular epithelium
decrease, while there is an increase in the stroma (Ghabreau et al.,
2004).
A recent study investigated the expression of class 1 HDACs and
two HATs in human endometrium (Krusche et al., 2007). The
HDAC13 were found to have constitutive mRNA expression
throughout the menstrual cycle, likewise the mRNA of one of the
HATs; p300/CBP-associated factor is also expressed in human endo-
metrium without cyclic changes. In contrast, mRNA expression of
another HAT; general control non-derepressible 5 was reduced
during secretory phase compared with the proliferative phase
(Krusche et al., 2007). The same study found that HDAC1, and
HDAC3 protein expression was constitutive throughout the menstrual
cycle, though HDAC1 protein expression was found to be highly vari-
able between individuals (Krusche et al., 2007). HDAC2 protein,
however, was found to be slightly but signicantly elevated during
the secretory phase (Krusche et al., 2007).
New developments
With epigenetic regulation seemingly involved in many of the pro-
cesses occurring in human endometrium during the menstrual cycle,
and some evidence for altered expression of epigenetic modulators
both during the menstrual cycle and in endometrial pathologies, it is
of interest to discover whether global epigenetic change occurs
during the menstrual cycle. Certainly, in rats, injection of estradiol
results in increased histone acetylation in the uterus (Libby, 1972;
Guo and Gorski, 1989) suggesting a role by which the ovarian
steroid hormones could act through chromatin alterations to effect
gene transcription. Likewise, it has been shown that HDAC inhibitor
treatment can enhance the proliferative and morphogenetic actions
of estrogen in mice (Gunin et al., 2005).
We recently began investigating global histone acetylation levels
during the menstrual cycle in the human endometrium, constructing a
preliminary prole of global histone acetylation levels during the men-
strual cycle. Little information has been available on histone acetylation
in the endometrium with ours being the rst study to report on levels of
histone acetylation and how they alter during the menstrual cycle. To
date, two studies have investigated the expression of epigenetic modu-
lators during the menstrual cycle, with a report on the expression of
class 1 HDACs (Krusche et al., 2007) and recently on the expression
of DNMTs (Yamagata et al., 2009), but how these results correlate
with the global histone acetylation or DNA methylation status of the
endometrium during the menstrual cycle has not yet been elucidated.
We found that global histone acetylation levels of H2AK5, H3K9
and H4K8 were increased in the early proliferative phase, sub-
sequently declining until ovulation, a trend shared by H3K14/18
(Figs 35). Histone acetylation is associated with transcription acti-
vation, so increased levels are consistent with the initiation of many
genes and pathways that would be required to regenerate the endo-
metrium following menstruation. Once regeneration has occurred
and proliferation has begun, it is expected that many of these pathways
are no longer required, so a subsequent decrease in acetylation levels
is not surprising. Likewise, the increase in acetylation post-ovulation
makes sense in light of the switch from proliferation to differentiation
by many of the cell types of the endometrium. This increase was stat-
istically signicant for H4K8 and a marked trend is observed for H3K9
and H4K14/18 which is supported by a previous study (Sakai et al.,
2003) that found that endometrial stromal cells cultured with estrogen
and progesterone exhibited a mild but signicant increase in the acety-
lation of H4K8 and H3K9/14. A decline in global acetylation levels in
the late secretory phase is also expected with the regression of the
corpus luteum and breakdown of the endometrium in the absence
of pregnancy. Global H2BK12 acetylation does not appear to share
the trend seen with the other acetylation sites during the proliferative
phase; however, the extent to which both H2A and H2B acetylation
contribute to transcriptional activation is less understood with most
studies focusing on histones H3 and H4.
H4K5 acetylation did not change during the menstrual cycle, although
this was expected as H4K5 (and H4K12; not examined in this study) is
associated with the deposition of newly transcribed histones onto
DNA, requiring the presence of histone chaperones (Verreault,
2000). The initial acetylation of histones following transcription is
thought to be important for their assembly into nucleosomes by
histone chaperones (Shahbazian and Grunstein, 2007). In many eukar-
yotes, newly synthesised histone H4 is acetylated at H4K5 and
H4K12 (Sobel et al., 1995). Hence, constant levels of H4K5 acetylation
could reect a maintained proportion of new histone when compared
with total histone throughout the menstrual cycle.
It is noted that these are preliminary data and not without limit-
ations. In particular, these data relate to whole endometrial tissue,
and therefore data relates to a mixed population of cells, the relative
mix of which will be changing somewhat during the menstrual cycle.
Experiments are continuing to address these concerns and to eluci-
date which changes result from alterations in steroid hormone
levels. In addition, as global bulk acetylation levels were examined,
data are not restricted to particular regions of the genome. Localized
Epigenetic regulation of human endometrium 303

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increases in acetylation may therefore have been masked in these
studies, and promoter-specic analyses (e.g. chromatin immunopreci-
pitation) will be required to determine whether this is the case.
Implications for future research/clinical
translation
Many studies have now examined the changes in endometrial gene
expression during the menstrual cycle both from freshly frozen
samples and using cell culture experiments (Popovici et al., 2000;
Brar et al., 2001; Carson et al., 2002; Kao et al., 2002; Borthwick
et al., 2003; Okada et al., 2003; Riesewijk et al., 2003;
Tierney et al., 2003; Ponnampalam et al., 2004; Mirkin et al., 2005;
Punyadeera et al., 2005; Talbi et al., 2006), often with little correlation
between studies. If epigenetic changes are indeed occurring during the
menstrual cycle then this could explain the large numbers of genes
showing differential expression across the cycle and between
studies. In particular, the effect of epigenetic alterations in cancer
cell lines must be considered when elucidating endometrial function,
with many studies to date using Ishikawa and other such cell lines
when examining events such as implantation.
Figure 4 Global acetylation of histone H3 at lysine 9 (A) and at
lysines 14 and 18 (B), in human endometrium during the menstrual
cycle. Changes in histone acetylation status were determined by
western blotting and data were normalized to histone H3 protein
levels and given as mean optical densities [relative to a sodium buty-
rate (HDAC inhibitor) treated positive control] +SEM. Statistical sig-
nicance denoted by: *P ,0.05. Cycle stages are early proliferative
[EP, (A) n 4, (B) n 5], mid-proliferative (MP, n 9), late prolif-
erative (LP, n 3), early secretory (ES, n 5), mid-secretory (MS,
n 4) and late secretory (LS, n 7) as determined by
histopathology.
Figure 3 Global acetylation of histones H2A (lysine 5) (A) and
H2B (lysine 12) (B), in human endometrium during the menstrual
cycle. Changes in histone acetylation status were determined by
western blotting and data were normalized to histone H2A/B
protein levels and given as mean optical densities [relative to a
sodium butyrate (HDAC inhibitor) treated positive control] +SEM.
Statistical signicance denoted by: **P ,0.01, ***P ,0.001. Cycle
stages are early proliferative [EP, (A) n 3, (B) n 4], mid-
proliferative (MP, n 9), late proliferative (LP, n 3), early secretory
(ES, n 5), mid-secretory (MS, n 4) and late secretory (LS, n 7)
as determined by histopathology. Numbers above bars indicate
P-values for non-signicant trends.
304 Munro et al.

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In addition, many studies into endometrial abnormalities compare
epigenetic state of the affected tissue/cells with normal endome-
trium. However, little regard is given to endometrial cycle stage,
with data invariably being pooled if no change is seen between the
proliferative and secretory phases. Given the apparent changes
within these phases reported here, much potential insight into these
disorders could be going unidentied.
Epigenetics appears to play a key role in developmental processes
that occur during the menstrual cycle and the establishment of preg-
nancy. It is potentially involved in the initial regeneration of the endo-
metrium through the changes in the epigenetic prole of stem cells and
is likely to be involved in endometrial proliferation and angiogenesis.
Evidence for altered epigenetic modulators or marks can be seen in
cancer and endometriosis, and epigenetics is also implicated in the
process of decidualization. Modulation of epigenetic state may there-
fore represent an important means of elucidating specic functions and
lead to therapeutic intervention.
Ethics
Ethical approval for the study was obtained from Northern X Ethics
Committee (NTX/08/02/008).
Acknowledgements
We thank the staff of Auckland City Surgical Services, Auckland City
Hospital and Greenlane Clinical Centre, Auckland, New Zealand for
their assistance in collection of the endometrial tissues, the clinicians
and patients involved, and the staff of Diagnostic Medlab for assistance
with histopathology of samples. We also thank Dr Kevin Dudley for
critical review of this manuscript.
Funding
This work was supported by the National Research Centre for
Growth and Development; a James Cook Research Fellowship to
MDM; and the Foundation for Research, Science and Technology
[grant# UOAX 0814].
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