mol. hum. reprod.-2010-munro-297-310

NEW RESEARCH HORIZON Review

Epigenetic regulation of endometriumduring the menstrual cycleS.K. Munro1,2, C.M. Farquhar3, M.D. Mitchell 1,2,4,and A.P. Ponnampalam1,*

1The Liggins Institute, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand 2The National Research Centre for Growthand Development, c/- The Liggins Institute, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand 3Department ofObstetrics and Gynaecology, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1142,New Zealand 4University of Queensland Centre for Clinical Research, RBWH Campus, Herston, Brisbane, QLD 4029, Australia

*Correspondence address. E-mail: [email protected]

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 uterinereceptivity. These changes are driven by estrogen and progesterone and involve the fine control of many different genes—several of whichhave been identified as being epigenetically regulated. Epigenetic modification may therefore influence the functional changes in the endo-metrium required for successful implantation. There is, however, only limited information on epigenetic regulation in endometrium. Wereview the potential role of epigenetic regulation of key processes during the menstrual cycle and present our own findings following a pre-liminary study into global acetylation levels in the human endometrium. A changing epigenetic state is associated with the differentiation ofstem 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 endometrialproliferation and differentiation. The processes of decidualization and implantation are also associated with epigenetic change and epigeneticmodulators show variable expression across the menstrual cycle. Our own studies found that endometrial global histone acetylation, asdetermined by western blotting, changed throughout the menstrual cycle and correlated well with expected transcription activity duringthe different phases. This suggests that epigenetics may be involved in the regulation of endometrial gene expression during the menstrualcycle and that abnormal epigenetic modifications may therefore be associated with implantation failure and early pregnancy loss as well aswith other endometrial pathologies.

Key words: endometrium / epigenetics / histone acetylation / menstrual cycle / uterus

IntroductionImplantation is a highly controlled process, involving a dialogue betweenthe endometrium and the implanting embryo which is crucial for theestablishment and maintenance of pregnancy (Norwitz et al., 2001;Dey et al., 2004; Makker and Singh, 2006; Pafilis et al., 2007). Little isknown 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). Controlof gene expression is crucial to the developmental processes which areregularly implemented as the endometrium undergoes cyclic periods ofgrowth and differentiation. Inappropriate epigenetic modification result-ing in aberrant expression of endometrial regulatory genes or proteinsmay therefore provide a partial explanation for the failure of embryoimplantation (Kao et al., 2003).

Deregulation of implantation may have consequences beyond thefailure of implantation and subsequent infertility. In mice, even a transi-ent postponement of blastocyst attachment is sufficient to cause a

detrimental ripple effect throughout the pregnancy, with aberrantspacing of embryos, defective placentation, resorption and retardeddevelopment of foetuses observed (Wilcox et al., 1999; Song et al.,2002; Ye et al., 2005). Poor implantation and placentation have alsobeen associated with end results such as intrauterine growth restriction,pre-eclampsia, and preterm birth in humans (Wang and Dey, 2006).

Epigenetic modification, resulting in altered chromatin structure andtranscriptional activity, controls gene expression and may thereforecontrol the functional changes in the endometrium which occurthroughout the menstrual cycle. Here, we examine the role epige-netics plays in several key processes which occur during the menstrualcycle and the establishment of pregnancy and discuss whether‘normal’ human endometrium could be under epigenetic regulation.

EpigeneticsFor mainly historical reasons, there has been some confusion aboutwhat ‘epigenetics’ actually refers to. A standard definition 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: [email protected]

Molecular Human Reproduction, Vol.16, No.5 pp. 297–310, 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 simplestsense; whereas other alterations seem to be heritable, but are notmaintained in a stable manner. These issues led to a proposed defi-nition of epigenetics as ‘the structural adaptation of chromosomalregions 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 withoutalterations in the DNA sequence’ (Berger et al., 2009).

Changes in chromatin architecture can be mediated by DNAmethylation and the post-translational modification of histone tails(Dolinoy et al., 2007; Jirtle and Skinner, 2007). These histone modifi-cations are also involved in the recruitment of transcription factorcomplexes (Berger, 2007) and therefore act to regulate geneexpression. It is thought that non-protein coding RNAs may also regu-late chromatin structure, with RNA mediated interplay between theenvironment, epigenome and transcriptome also occurring, howeverthis is still an emerging area of research (Mattick et al., 2009). Cellularcomponents such as DNA methyltransferases (DNMTs), histone dea-cetylases (HDACs) and histone acetyltransferases (HATs) act to main-tain or alter these modifications as appropriate. Two of the mostwell-known epigenetic-related phenomena are X chromosome inacti-vation and genomic imprinting (Jirtle and Skinner, 2007). A range ofenvironmental factors, including nutrition and exposure to xenobioticchemicals, can influence 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 modifications have been foundto be altered in human cancers within the promoter regions of tumoursuppressor genes and oncogenes (Jirtle and Skinner, 2007). It isbecoming increasingly clear that such changes are also associatedwith other disease states (Guo, 2009; Trenkmann et al., 2009;Turunen et al., 2009).

DNA methylationDNA methylation is the covalent modification of post-replicative DNAby the addition of a methyl group to the cytosine ring to form methylcytosine; 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 methylationafter replication, and DNMTs 3a and 3b, which predominantly func-tion as de novo methyltransferases acting at previously un-methylatedsites (Brero et al., 2006).

In mammals, DNA methylation is thought to be exclusively associ-ated with cytosine-phosphate-guanine (CpG) dinucleotides, occurringon both strands at the cytosine residue (Brero et al., 2006). In ver-tebrates, large un-methylated GC rich regions can be found at the50 end of many genes and are termed CpG islands (Bird, 1987).DNA methylation at promoter associated CpG islands is stronglylinked to repression of transcriptional activity; however, the preciserole of CpG methylation is an area of ongoing research (Bird,2002). Approximately 10–20% of genes display DNA methylationpatterns in a tissue-specific manner (Song et al., 2005; Eckhardtet al., 2006; Rakyan et al., 2008) and these so-called tissue-specific dif-ferentially methylated regions have been associated with tissue-specific

patterns of gene expression (Rakyan et al., 2008). The repressivenature of DNA methylation is thought to result from either themethyl group inhibiting the binding of regulatory molecules to aCpG site or by acting as a recognition signal for methyl-CpG-bindingproteins with repressive properties (Bird, 2002), both of which aremechanisms likely to act in a site-specific manner. However, itshould be noted that DNA methylation is not always associatedwith transcriptional repression. A recent study confirmed previousfindings of a negative correlation between DNA methylation andgene expression at promoters with a medium or high CpG density,however, contrary to previous notions, even some low-CpG densitypromoters showed this correlation. In contrast, gene-body methyl-ation was found to positively correlate with gene expression suggestingnovel roles for DNA methylation (Rakyan et al., 2008). Furtherresearch is required to elucidate the sequence and context-specificnature of this epigenetic modification.

Histone modificationsHistones are integral to the higher order structure of chromatin.Approximately 146 bp of DNA is wrapped around a histoneoctamer consisting of two sets of the core histones—H2A, H2B,H3 and H4—to form a nucleosome. These nucleosomes are linkedby loops of DNA and the linker histone H1. Although the globulardomains of histones associate closely with each other, the trailingamino acid ‘tails’ protrude past the surrounding DNA and aresubject to post-translational modification. New histone proteins areproduced during the S (synthesis) phase of the cell cycle whenDNA 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 thehistones, synthesized de novo, are then deposited (Sogo et al.,1986). Two H3–H4 heterodimers are deposited either sequentiallyor as a heterotetramer, followed by the two H2A–H2B dimers,thus forming the octamer (Worcel et al., 1978; English et al., 2006).

Many distinct modifications of histones exist, with those identifiedincluding lysine acetylation, lysine methylation, arginine methylation,phosphorylation, ubiquitylation, sumoylation, ADP ribosylation, deimi-nation and proline isomerization (Kouzarides, 2007; Lindner, 2008).Modification of histones can be difficult to study as histone modifi-cations are very dynamic. All known histone post-translational modifi-cations have now been shown to be reversible, and as some histonemodifications have been shown to change within minutes of a stimulus,it is uncertain as to which modifications can be considered as trulystable and therefore epigenetic. It is also unknown how the persist-ence of chromatin state is achieved, and which modifications aretherefore truly heritable (Berger, 2007; Kouzarides, 2007).

Histone deacetylation, one of the best characterized histone modi-fications, is associated with gene silencing, whereas histone acetylationis associated with transcriptional activation (Fuks, 2005) (Fig. 1).Histone acetylation patterns are maintained by two opposing groupsof enzymes, the HATs and HDACs (Hildmann et al., 2007). Thereis also evidence for crosstalk between elements of the DNA methyl-ation machinery and those involved in histone modification, withDNMTs able to recruit complexes containing HDACs (Fuks, 2005).A group of proteins with methyl binding domains are also able torecognize methylated DNA sequences and recruit HDACs (Nanet al., 1998; Irvine et al., 2002; Jorgensen and Bird, 2002). A reverse

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regulatory pathway whereby histone acetylation mediates DNAdemethylation may also exist (Szyf et al., 1985; Selker, 1998;Cervoni and Szyf, 2001) as some data show that HDAC inhibitioncan 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 isnot always the case (Cameron et al., 1999; Singal et al., 2001;El-Osta et al., 2002). HDACs and HATs, like many other enzymesinvolved in histone modification, have actions which are not specificto the histones and are able to modify other proteins. Over 60 tran-scription factors including the tumour suppressor p53 are subjected toacetylation and some HATs also physically associate with non-proteincoding RNA (Yang and Seto, 2007). Similar actions are seen withother histone modifying enzymes, such as lysine-specificdemethylase-1, which in addition to histone demethylation, hasbeen found to be involved in the demethylation and stabilization ofDNMT1, which is required for the maintenance of global DNAmethylation (Hotz and Peters, 2009; Wang et al., 2009).

The menstrual cycleHuman endometrium undergoes cyclic morphological changes invol-ving precise periods of growth, differentiation and regression, witheach new menstrual cycle starting again on average every 28 daysduring a woman’s reproductive years (Curry and Osteen, 2003).These changes are the visible result of the interactions of an arrayof biological communication pathways and many local factors includingcytokines, which are under the overall control of the ovarian steroidsestrogen and progesterone (Curry and Osteen, 2003). Rising serumestrogen levels from the ovary stimulate endometrial growth duringthe proliferative phase. Epithelial and stromal cells from the functional

layer proliferate extensively, with both cell types increasingly acquiringreceptors to both estrogen and progesterone in parallel to the rise inestrogen 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 serumprogesterone levels produced by the corpus luteum (Brenner andWest, 1975; Lessey, 2000). Changes in the glandular epithelium areapparent by cycle Days 15–16 (Noyes et al., 1950; Lessey, 2000). Fol-lowing fertilization, signals from the developing blastocyst act inconcert with steroid hormones to induce a receptive state supportiveof implantation (Paria et al., 2002; Makker and Singh, 2006). The endo-metrial epithelium only transiently acquires this receptive state and avariety of architectural, cellular, molecular and biochemical eventsmust be coordinated (Makker and Singh, 2006). The endometrialstroma becomes more vascular and oedematous during this phase,with the glands displaying enhanced secretory activity (Norwitzet al., 2001). Additional morphological changes occur with theprocess of decidualization by which stromal cells differentiate tosupport 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 knownas menstruation (Carson et al., 2000). As menstruation results inbleeding and tissue loss, it is followed by a period of repair and regen-eration of the functionalis prior to the next cycle of proliferation. Theregenerative capacity of the endometrium is such that within a cycle itgrows to a thickness of 5–7 mm from the initial 0.5–1 mm post-menstruation (McLennan and Rydell, 1965). Consequently, theendometrium requires a continuing activation of processes such asangiogenesis 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 modifications (PTMs) influence chromatin structure. This leads to either a more condensed state, associatedwith transcriptional repression or a more relaxed state associated with transcriptional activation. PTMs are the net result from activity of epigeneticmodulators which form large complexes with some adding, others removing different modifications. Chromatin state is also linked to the degree ofDNA methylation with crosstalk between elements of the DNA methylation machinery and those involved in histone modification.

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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 endometriumThe stratum basalis or basal layer of the endometrium remains intactthroughout the menstrual cycle and contains the cells from which thefunctional 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 theremight be a local population of adult stem cells contributing to theregeneration of the functional layer (Padykula, 1991) (Fig. 2). Adultstem cells are small populations of quiescent cells that undergo asym-metrical cell divisions when activated, allowing the maintenance oftheir own population in addition to the production of daughtercells—like embryonic stem cells—are likely to be under the controlof DNA methylation and chromatin modifications. These adult stemcells/progenitor cells can proliferate extensively (Fuchs and Segre,2000) differentiating to form the different cell types of that lineageand represent a more differentiated version of the stem cells seenin early development.

It is clear that there are major epigenetic alterations of the genomeduring mammalian development and during differentiation of embryo-nic stem cells (Reik, 2007). Embryonic stem cells differ in their globalgene expression profile from the more lineage restricted adult stemcells, with a gradual loss of pluripotent gene expression and gain oflineage-specific 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 profiles and thus stem cell iden-tity, with the epigenetic ‘code’ determining a stem cell’sresponsiveness to developmental signals (Cirillo et al., 2002; Schremet al., 2002; Zaret et al., 2008; Collas, 2009). External stimuli suchas 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 propertiesof stem/progenitor cells, pluripotent differentiation and self-renewalseem to be under the control of DNA methylation and chromatinmodifications (Shukla et al., 2008). In light of this, it is not surprisingthat alteration of epigenetic state has been found to play an importantrole in the reprogramming of cell fate, with studies showing that vir-tually any cell can be regressed to a less mature state with the rightcombination 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 alteredepigenetic patterns in tissue stem/progenitor cells (Feinberg et al.,2006) emphasizing the importance of epigenetics in control of cellproliferation and differentiation.

The first endometrial stem/progenitor cell candidates were ident-ified in 2004, with a small population of both human endometrial epi-thelial cells and stromal cells shown to possess clonogenic activityin vitro (Chan et al., 2004). It has been suggested that these colony-forming unit (CFU) cells might be responsible for the regenerationof both cycling and atrophic endometrium (Gargett et al., 2008),with CFU cells being found in normal cycling endometrium, inactiveperi-menopausal endometrium and in the endometrium of womentaking oral contraceptives (Schwab et al., 2005). The existence of epi-thelial and stromal stem/progenitor cells in human and mouse endo-metrium have been confirmed using stem cell activity assays (Gargettet al., 2007; Gargett et al., 2009). Assays also recently identified differ-ent CFU cells as being of an epithelial progenitor cell type (Gargettet al., 2009), which is expected to reside somewhere in the basalislayer (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 nichesin both the basal and functional layers of the endometrium (Schwaband Gargett, 2007). Epithelial side population cells have alsobeen identified 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 processesof stem cell differentiation, angiogenesis, implantation and decidualization, and it is likely that given the dynamic nature of the endometrium, that it isunder epigenetic regulation.

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(Kato et al., 2007; Tsuji et al., 2008) and are reported to be capable ofreconstituting various endometrial components and structures whenxenotransplanted into mice (Maruyama, 2009).

It has been proposed that inappropriate shedding of endometrialprogenitor cells may contribute to the development of endometrioticlesions, with evidence of monoclonality (Jimbo et al., 1997; Starzinski-Powitz et al., 2001; Leyendecker et al., 2002; Tanaka et al., 2003; Wuet al., 2003; Gargett, 2007; Gargett et al., 2008). With a change inenvironment following retrograde menstruation potentially alteringboth paracrine and endocrine signalling, it is likely that a number ofepigenetic changes arise in ectopic endometrium, consequentlygiving rise to the pathology of endometriosis. Indeed, endometriosisis increasingly being identified as an epigenetic disease and the evi-dence for this has recently been reviewed (Guo, 2009). Evidence ofdistinct epigenetic changes arising in endometriosis suggests a mechan-ism by which key endometrial genes involved in proliferation anddifferentiation may already be under epigenetic control, with acti-vation/repression in response to environmental cues. It is likely thatepigenetic changes assist in the regulation of progenitor cell differen-tiation into the variety of lineage-restricted endometrial cell typesrequired 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 migrateover the denuded surface of the endometrium (Okulicz and Scarrell,1998). At this time, endometrial epithelial cells also lack theexpression of estrogen receptor alpha (ERa). A mouse model ofendometrial restoration following menstruation has confirmed regen-eration can occur in the absence of estrogen (Kaitu’u-Lino et al.,2007). Rapid proliferation of epithelial label-retaining cells in responseto estrogen despite the lack of receptor indicates a transmission ofproliferative signals from stromal niche cells (which do express ERa)to candidate stem/progenitor cells (Chan and Gargett, 2006;Gargett, 2007) and exemplifies the importance of local environmentin the control of endometrial repair and regeneration.

ProliferationDuring the proliferative phase, rising serum estrogen levels from theovary stimulate endometrial growth with extensive proliferation ofthe epithelial and stromal cells of the functional layer, which increas-ingly acquire receptors to both estrogen and progesterone (Garciaet al., 1988). Studies into endometrial cancer have allowed us someinsight into the potential for epigenetic regulation of normal endo-metrial proliferation. For example promoter hypo-methylation of thetranscription factor paired-box gene 2 (PAX2) has been identified inendometrial cancer, with 75% of endometrial carcinoma in onestudy exhibiting PAX2 promoter hypo-methylation (Wu et al.,2005a). This hypo-methylation was found to allow inappropriateactivation by estrogen and tamoxifen, and resulted in enhanced cellproliferation (Wu et al., 2005a). This suggests that endometrialresponsiveness to estrogen signalling may be dependent not only onreceptor expression but also on the appropriate epigenetic modifi-cation of other effectors. Receptiveness to steroid hormones andtheir antagonists is a particular problem with endometrial tissue, par-ticularly in patients receiving treatment for breast cancer. Tamoxifen, aselective ER modulator which acts as an ER antagonist in the breastand is used for breast cancer therapy, acts as a partial agonist in theendometrium (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 blockthe proliferation of uterine endometrial cells exposed to both tamox-ifen and estrogen in Ishikawa cells (Hodges-Gallagher et al., 2007). TheIshikawa 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.

AngiogenesisAngiogenesis is the process of new blood vessel growth and occurs reg-ularly during every menstrual cycle in human endometrium, with thegrowth and regression of blood vessels occurring under the influenceof estrogen and progesterone. The regulation of angiogenesis in theendometrium has been difficult to elucidate, with a large number ofangiogenic factors being secreted. The direct correlation of thesefactors with endometrial angiogenesis is hard to confirm as endothelialcell proliferation, a marker for angiogenic activity, occurs throughoutthe menstrual cycle (Gargett and Rogers, 2001; Rogers et al., 2009). Inanimal models, endothelial cell proliferation is closely linked to circulatinglevels of estrogen and progesterone; however, regulation is not so simplein humans, with evidence that estrogen can both promote and inhibitangiogenesis under different circumstances (Girling and Rogers, 2005).

In mouse models, endothelial cell proliferation occurs following theelevation of estrogen levels post-estrus, and in conjunction with risingplasma progesterone levels (Walter et al., 2005). In ovariectomizedmice, this proliferation can be induced to occur within 24 hours byinjection with 100 ng of estrogen 7 days post-surgery (Heryanto andRogers, 2002). Treatment with either antibodies against vascularendothelial growth factor (VEGF) or VEGF receptor 2 inhibitors, com-pletely blocks this response (Heryanto et al., 2003), suggesting thatestrogen acts through the VEGF pathway. An influx of VEGF expres-sing neutrophils into the endometrium also occurs following estrogeninjection, which when blocked, results in decreased proliferation ofendothelial 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 stimulatedendothelial cell proliferation, although this effect is only mediated inpart through the VEGF pathway (Walter et al., 2005).

In the human endometrium, VEGF is the principle angiogenic factorand its action is not restricted to vascular smooth muscle cells(Charnock-Jones et al., 1993). VEGF mRNA is expressed mainly inthe stroma during the proliferative phase (Li et al., 1994; Kawanoet al., 2000), with a subset of stromal cells exhibiting much higherexpression, and a low level of expression is also observed in the gland-ular epithelium (Charnock-Jones et al., 1993). During the secretoryphase stromal VEGF expression is more uniform and glandularexpression is increased (Charnock-Jones et al., 1993), with overallexpression three to five times higher in the secretory phase whencompared with the early proliferative phase (Charnock-Jones et al.,1994). A high level of VEGF mRNA can also be detected in menstrualtissue (Charnock-Jones et al., 1993). Focal VEGF is also located in neu-trophils associated with micro-vessel walls (Gargett et al., 2001). Instromal cells, VEGF production is accompanied by the production ofmatrix metalloproteinases (MMPs) which are involved in extracellularmatrix degradation and regulation of angiogenesis (Li et al., 1994;




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Moses and Langer, 1991; Kawano et al., 2000). Treatment withthrombin leads to increased VEGF, MMP-1 and active MMP-2, andthis up-regulation appears to act through PAR-1 (a member of theG-protein-coupled protease-activated receptors) and mitogen-activated protein kinase (Furukawa et al., 2009).

Evidence from other tissues suggests that epigenetic mechanisms playa role in the process of angiogenesis. For example, studies have shownthat VEGF actions on endothelial cell proliferation are mediated throughregulation 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 conservedserine residues in the N-terminal domain result in the removal of thisclass of HDACs from the nucleus to the cytoplasm, activating geneexpression (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-10expression in endothelial cells and in vitro assays support a role forthe modulation of angiogenesis by HDAC-7 (Chang et al., 2006;Mottet et al., 2007). Inhibition of HDAC-7 gene transcription resultsin altered endothelial cell morphology, migration and capillary formingcapacity 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 ofHDACs blocks the differentiation of endothelial progenitor cells andthis appears to act through the down-regulation of homeoboxprotein HOXA9, as over expression of HOXA9 partially rescues thedifferentiation blockade observed after HDAC inhibitor treatment(Rossig et al., 2005). HOXA9 appears to act as a regulator ofendothelial-committed genes such as endothelial nitric oxide synthase,VEGFR-2 and VE-cadherin, and also mediates the shear stress-inducedmaturation of endothelial cells (Rossig et al., 2005). This implicateshistone acetylation in the control of angiogenesis, with VEGF signallingmediated through HDAC-7 resulting in the up-regulation of a numberof genes critical to angiogenesis, and it is possible that similar mechan-isms may be acting in the endometrium (Fig. 2). HOXA9 mRNA isexpressed in human endometrium with a decrease in expressionreported between the early and mid-secretory phase (Carson et al.,2002). Interestingly, the HOXA9 gene in endometrium has beenshown to be methylated in women with ovarian cancer, which isthought to result from abnormal methylation of HOXA9 in mulleriantract stem cells (Widschwendter et al., 2009). PDGF-B transcriptshave been identified in human endometrium (Boehm et al., 1990),and both PGDF-B and MMP10 are highly expressed by a populationof cells isolated from menstrual blood termed ‘endometrial regenerativecells’ (ERC) which were capable of differentiation into a number oflineages including epithelial and endothelial lineages (Meng et al.,2007). In addition, MMP10 production by endometrial stromal cellshas been shown to increase in response to conditioned media fromhuman trophoblasts (Hess et al., 2007), which may assist in vascularremodelling associated with placentation.

DifferentiationRising serum progesterone levels post-ovulation result in a secretorytransformation 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 Ishikawacells are a well-differentiated adenocarcinoma cell line rather than a‘normal’ endometrial cell line type (Nishida, 2002), treatment withthe related HDAC inhibitors Trichostatin A (TSA) and suberoylanilidehydroxamic acid (SAHA), results in differentiation which closelyresemble normal secretory phase endometrial epithelium in a time-and dose-dependent manner. This is accompanied by the expressionof secretory phase-specific proteins (Uchida et al., 2005b) and theeffects on proliferation and differentiation by TSA and SAHA werecomparable to treatment with estrogen and progesterone suggestinga parallel between histone hyper-acetylation, differentiation andsecretory phase hormone exposure. Surprisingly, the HDAC inhibitoreffects could be blocked by gene silencing of glycodelin, an establisheddifferentiation marker for endometrial glandular cells (Seppala et al.,2002) which suggests some level of feedback.

Decidualization is a process of differentiation of the fibroblast-likestromal cells of the endometrium and is necessary to support thedeveloping embryo (Sakai et al., 2003). These cells, primed by estro-gen in the proliferative stage, respond to progesterone by becominglarger and rounder and initiating a range of biochemical pathways.Decidual cells arise first in the vicinity of the spiral arteries, andbegin to spread throughout the endometrium during the secretoryphase 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 epigeneticchanges in vitro (Fig. 2). HDAC inhibitor treatment of human endo-metrial stromal cells with TSA resulted in morphological change andexpression of differentiation marker proteins such as insulin-likegrowth factor (IGFBP-1) and prolactin, indicative of decidualization(Sakai et al., 2003). It was found that histones H3 and H4 becameacetylated 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 adenocarcinomacell lines treated with TSA or SAHA have been shown to undergomorphological transformation to a flattened and widespread epithelialphenotype, with induction of differentiation markers such as glycode-lin, leukaemia inhibitory factor (LIF), interleukin-1 receptor and glyco-gen synthesis—similar to responses seen with ovarian steroidhormones (Uchida et al., 2005a).

ImplantationWith epigenetics implicated in the control of decidualization (Sakaiet al., 2003; Uchida et al., 2007b), it is likely that epigenetic regulationplays a role in the establishment of endometrial receptivity and in sub-sequent embryo implantation (Fig. 2). DNA methylation and histoneacetylation have been directly correlated with the expression ofimplantation related genes in other contexts, including LIF (Uchidaet al., 2005b), HOXA10 (Wu et al., 2005b; Yoshida et al., 2006), gly-codelin (Uchida et al., 2005b; Uchida et al., 2007a), MMPs, tissueinhibitors 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

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epigenetic modulation with promoter associated CpG islands(Campan et al., 2006) and are capable of inducing the modificationto the chromatin structure of other genes (Leu et al., 2004). In turnthese epigenetically modified genes are involved in the expression ofMUC1 (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 hasbeen linked to the formation of pinopodes (Bagot et al., 2001;Aghajanova et al., 2003), with many secondary interactions betweenthese factors. It is conceivable that many other genes involved aresimilarly regulated.

Expression of epigenetic modulators duringthe menstrual cycleSignificant changes in the expressions of DNMTs have been observedin endometrium during the menstrual cycle with a reported decreasein DNMT mRNA during mid-late secretory phase, and followingestrogen and medroxy-progesterone acetate treatment in stromalcell cultures (Yamagata et al., 2009). DNMT protein levels in humanendometrium remain less clear, with reports of no change inDNMT expression during the menstrual cycle by one group (Yamagataet 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 incells of the glandular epithelium and the stroma. In the secretory phaselevels of methylated cytosines in the cells of the glandular epitheliumdecrease, while there is an increase in the stroma (Ghabreau et al.,2004).

A recent study investigated the expression of class 1 HDACs andtwo HATs in human endometrium (Krusche et al., 2007). TheHDAC1–3 were found to have constitutive mRNA expressionthroughout the menstrual cycle, likewise the mRNA of one of theHATs; p300/CBP-associated factor is also expressed in human endo-metrium without cyclic changes. In contrast, mRNA expression ofanother HAT; general control non-derepressible 5 was reducedduring secretory phase compared with the proliferative phase(Krusche et al., 2007). The same study found that HDAC1, andHDAC3 protein expression was constitutive throughout the menstrualcycle, 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 significantly elevated duringthe secretory phase (Krusche et al., 2007).

New developmentsWith 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 modulatorsboth during the menstrual cycle and in endometrial pathologies, it isof interest to discover whether global epigenetic change occursduring the menstrual cycle. Certainly, in rats, injection of estradiolresults in increased histone acetylation in the uterus (Libby, 1972;Guo and Gorski, 1989) suggesting a role by which the ovariansteroid hormones could act through chromatin alterations to effectgene transcription. Likewise, it has been shown that HDAC inhibitor

treatment can enhance the proliferative and morphogenetic actionsof estrogen in mice (Gunin et al., 2005).

We recently began investigating global histone acetylation levelsduring the menstrual cycle in the human endometrium, constructing apreliminary profile of global histone acetylation levels during the men-strual cycle. Little information has been available on histone acetylationin the endometrium with ours being the first study to report on levels ofhistone acetylation and how they alter during the menstrual cycle. Todate, two studies have investigated the expression of epigenetic modu-lators during the menstrual cycle, with a report on the expression ofclass 1 HDACs (Krusche et al., 2007) and recently on the expressionof DNMTs (Yamagata et al., 2009), but how these results correlatewith the global histone acetylation or DNA methylation status of theendometrium during the menstrual cycle has not yet been elucidated.

We found that global histone acetylation levels of H2AK5, H3K9and H4K8 were increased in the early proliferative phase, sub-sequently declining until ovulation, a trend shared by H3K14/18(Figs 3–5). Histone acetylation is associated with transcription acti-vation, so increased levels are consistent with the initiation of manygenes and pathways that would be required to regenerate the endo-metrium following menstruation. Once regeneration has occurredand proliferation has begun, it is expected that many of these pathwaysare no longer required, so a subsequent decrease in acetylation levelsis not surprising. Likewise, the increase in acetylation post-ovulationmakes sense in light of the switch from proliferation to differentiationby many of the cell types of the endometrium. This increase was stat-istically significant for H4K8 and a marked trend is observed for H3K9and H4K14/18 which is supported by a previous study (Sakai et al.,2003) that found that endometrial stromal cells cultured with estrogenand progesterone exhibited a mild but significant increase in the acety-lation of H4K8 and H3K9/14. A decline in global acetylation levels inthe late secretory phase is also expected with the regression of thecorpus luteum and breakdown of the endometrium in the absenceof pregnancy. Global H2BK12 acetylation does not appear to sharethe trend seen with the other acetylation sites during the proliferativephase; however, the extent to which both H2A and H2B acetylationcontribute to transcriptional activation is less understood with moststudies focusing on histones H3 and H4.

H4K5 acetylation did not change during the menstrual cycle, althoughthis was expected as H4K5 (and H4K12; not examined in this study) isassociated with the deposition of newly transcribed histones ontoDNA, requiring the presence of histone chaperones (Verreault,2000). The initial acetylation of histones following transcription isthought to be important for their assembly into nucleosomes byhistone chaperones (Shahbazian and Grunstein, 2007). In many eukar-yotes, newly synthesised histone H4 is acetylated at H4K5 andH4K12 (Sobel et al., 1995). Hence, constant levels of H4K5 acetylationcould reflect a maintained proportion of new histone when comparedwith 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 relativemix 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 hormonelevels. In addition, as global bulk acetylation levels were examined,data are not restricted to particular regions of the genome. Localized




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increases in acetylation may therefore have been masked in thesestudies, and promoter-specific analyses (e.g. chromatin immunopreci-pitation) will be required to determine whether this is the case.

Implications for future research/clinicaltranslationMany studies have now examined the changes in endometrial geneexpression during the menstrual cycle both from freshly frozensamples and using cell culture experiments (Popovici et al., 2000;

Brar et al., 2001; Carson et al., 2002; Kao et al., 2002; Borthwicket 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 correlationbetween studies. If epigenetic changes are indeed occurring during themenstrual cycle then this could explain the large numbers of genesshowing differential expression across the cycle and betweenstudies. In particular, the effect of epigenetic alterations in cancercell lines must be considered when elucidating endometrial function,with many studies to date using Ishikawa and other such cell lineswhen examining events such as implantation.

Figure 4 Global acetylation of histone H3 at lysine 9 (A) and atlysines 14 and 18 (B), in human endometrium during the menstrualcycle. Changes in histone acetylation status were determined bywestern blotting and data were normalized to histone H3 proteinlevels and given as mean optical densities [relative to a sodium buty-rate (HDAC inhibitor) treated positive control]+ SEM. Statistical sig-nificance denoted by: *P , 0.05. Cycle stages are early proliferative[EP, (A) n ¼ 4, (B) n ¼ 5], mid-proliferative (MP, n ¼ 9), late prolif-erative (LP, n ¼ 3), early secretory (ES, n ¼ 5), mid-secretory (MS,n ¼ 4) and late secretory (LS, n ¼ 7) as determined byhistopathology.

Figure 3 Global acetylation of histones H2A (lysine 5) (A) andH2B (lysine 12) (B), in human endometrium during the menstrualcycle. Changes in histone acetylation status were determined bywestern blotting and data were normalized to histone H2A/Bprotein levels and given as mean optical densities [relative to asodium butyrate (HDAC inhibitor) treated positive control]+ SEM.Statistical significance denoted by: **P , 0.01, ***P , 0.001. Cyclestages are early proliferative [EP, (A) n ¼ 3, (B) n ¼ 4], mid-proliferative (MP, n ¼ 9), late proliferative (LP, n ¼ 3), early secretory(ES, n ¼ 5), mid-secretory (MS, n ¼ 4) and late secretory (LS, n ¼ 7)as determined by histopathology. Numbers above bars indicateP-values for non-significant trends.

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In addition, many studies into endometrial abnormalities compareepigenetic 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 theproliferative and secretory phases. Given the apparent changeswithin these phases reported here, much potential insight into thesedisorders could be going unidentified.

Epigenetics appears to play a key role in developmental processesthat 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 profile of stem cells and

is likely to be involved in endometrial proliferation and angiogenesis.Evidence for altered epigenetic modulators or marks can be seen incancer and endometriosis, and epigenetics is also implicated in theprocess of decidualization. Modulation of epigenetic state may there-fore represent an important means of elucidating specific functions andlead to therapeutic intervention.

EthicsEthical approval for the study was obtained from Northern X EthicsCommittee (NTX/08/02/008).

AcknowledgementsWe thank the staff of Auckland City Surgical Services, Auckland CityHospital and Greenlane Clinical Centre, Auckland, New Zealand fortheir assistance in collection of the endometrial tissues, the cliniciansand patients involved, and the staff of Diagnostic Medlab for assistancewith histopathology of samples. We also thank Dr Kevin Dudley forcritical review of this manuscript.

FundingThis work was supported by the National Research Centre forGrowth and Development; a James Cook Research Fellowship toMDM; and the Foundation for Research, Science and Technology[grant# UOAX 0814].

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