American Journal of Obstetrics and Gynecology (2005) 193, 371–80

www.ajog.org

Aberrant methylation at HOXA10 may be responsible forits aberrant expression in the endometrium of patientswith endometriosis

Yan Wu, PhD,a Gloria Halverson, MD,b Zainab Basir, MD,c Estil Strawn, MD,b

Pearlly Yan, PhD,d Sun-Wei Guo, PhDa,*

Departments of Pediatrics,a Obstetrics and Gynecology,b Pathology,c Medical College of Wisconsin, Milwaukee, WI;and Division of Human Cancer Genetics, Department of Molecular Virology, Immunology, and Medical Genetics,Comprehensive Cancer Center, The Ohio State University, Columbus, OH d

Received for publication August 4, 2004; revised December 31, 2004; accepted January 14, 2005

KEY WORDSEndometriosisEpigeneticsGene expression

HOXA10Methylation

Objective: HOXA10, expressed in endometrium, plays an important role in uterine receptivity atthe time of implantation. In the endometrium of women with endometriosis, its expression is

reduced. The aim of this study was to determine whether the observed aberrant expression ofHOXA10 is caused by aberrant methylation of the gene.Study design: Endometrial tissues were collected from 6 women with laparoscopically confirmed

endometriosis, and 4 women who underwent tubal ligation and were confirmed to have noendometriosis. In addition, menstrual blood from 5 women with no gynecologic complaints wascollected and also used as controls. Methylation-specific polymerase chain reaction (PCR) andbisulfite sequencing were performed in 3 fragments in 2 regions of HOXA10 to detect difference

in methylation patterns. Real-time reverse transcriptase (RT)-PCR was also performed tomeasure expression levels of HOXA10 in select cases and controls.Results: In all 3 fragments, there were highly statistically significant differences in methylation

patterns between women with endometriosis and those without endometriosis. The expressionlevel of HOXA10 was lower in women with endometriosis than those without, as previouslyreported.

Conclusion: There is aberrant methylation in the endometrium of women with endometriosiscompared with those without endometriosis. The aberrant methylation at HOXA10 may beresponsible for the aberrant gene expression in the endometrium of women with endometriosis.

This finding suggests that endometriosis may also be an epigenetic disease.� 2005 Elsevier Inc. All rights reserved.

Supported in part by the Children’s Hospital of Wisconsin

Foundation.

* Reprint requests: Sun-Wei Guo, PhD, Dept of Pediatrics,

Medical College of Wisconsin, 8701 Watertown Plank Road, MS

756, Milwaukee, WI 53226-0509.

E-mail: swguo@mail.mcw.edu

0002-9378/$ - see front matter � 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.ajog.2005.01.034

There is mounting evidence suggesting that theeutopic endometrium in women with endometriosishas various alterations compared with those fromwomen without endometriosis.1 Recent work basedon large-scale expression microarrays indicates that,compared with normal controls, the endometrium

372 Wu et al

Figure 1 The transcript structure of human HOXA10 gene. This gene has several CpG islands within 50 upstream region of exon1and intron region. Three fragments within CpG islands (F1, F2, and F3) were sequenced after bisulfite treatment.

from women with endometriosis has aberrant expressionprofiles.2 We have recently shown that the endometriumfrom women with endometriosis contains considerablecryptic genomic alterations,3 which may be related to, oreven responsible for, genomic alterations in the ectopicendometrium4 and, hence, may be a proximate cause forendometriosis.

These reported aberrant alterations in the endome-trium from women with endometriosis, be it molecular,cellular, or transcriptional or structural, beg for anobvious question: what are the proximate causes thatare responsible for these alterations? Indeed, withoutany change in structure or cell memory, the maintenanceof all these alterations, be it transcriptional or bio-chemical, could be costly.

Epigenetic changes, or heritable changes in geneexpression patterns mediated by mechanisms otherthan DNA sequence alterations, especially methylations,can be a flexible yet stable mechanism for suppressingtranscriptional activities of a gene and have been welldemonstrated to be central to the neoplastic process.5,6

Recently, it has been demonstrated experimentally thataberrant methylations promote chromosomal instabilityand induce tumor.7,8 Despite its important role indevelopment and health,9,10 however, the role of aber-rant methylation, if any, in the pathogenesis of endo-metriosis so far has received scant attention.

HOXA10 is a member of a gene family which containsa common, conserved region of 183 bp, called homeo-box.11 The gene family functions as transcription factorsthat regulate a plethora of other genes in development. Inaddition to its role in uterine development,12 HOXA10expression has been shown to be important for uterine

receptivity to implantation in mice.13 In humans,HOXA10 has been shown to be expressed in endome-trium, and to be regulated by estrogen and progester-one.14,15 Its peak expression occurs during the windowof implantation, suggesting a possible role in uterinereceptivity. In women with endometriosis, which isknown to be associated with subfertility,16 it has beenshown that HOXA10 is aberrantly down-regulated intheir endometrium during the secretory phase comparedwith the control eutopic endometrium.17 The cause forthis alteration still remains unknown. In this study, weinvestigate patterns of methylations in the transcrip-tional start sites of HOXA10.

Material and methods

Tissue collection

Endometrial tissue samples were collected by pipellesuction curettage from 6 women, aged 33 to 36 years,with surgically and histologically confirmed stagesIII-IV endometriosis. For controls, endometrial biopsieswere obtained from 4 women undergoing tubal liga-tions, aged 26 to 36 years, without endometriosis,confirmed at laparoscopy. In addition, endometrialtissues were isolated from the menstrual blood of 5healthy women, aged 30 to 38 years, without anygynecologic complaints. The phase of menstrual cyclewas determined according to the criteria of Noyeset al.18 The endometrial tissue samples obtained fromwomen with endometriosis and women who underwenttubal ligation were snap frozen on dry ice immediatelyafter surgical dissection, and then stored in a �80(C

Wu et al 373

Figure 2 Partial methylation in the F2 fragment of HOXA10 by MSP with bisulfite-treated endometrial tissue samples fromendometriosis patients, normal controls, and normal lymphocytes. U, reactions using the HOXA10 primer set specific for the

unmethylated CpG sites giving rise to a 260-bp band. M, reactions using the primer set specific for the methylated CpG sites givingrise to a 260-bp band. WT, unmodified/wild-type. Mt is a molecular marker. (A) Lanes 1 to 12 were bisulfite-treated DNA frompatients with endometriosis, while lanes 13 to 18 were those from normal controls. Notice the very faint bands in the M lanes of allendometriosis patients and 2 controls, suggesting partial methylation. (B) Lanes 1 to 10 were bisulfite-treated DNA from menstrual

debris of healthy women. Notice the absence of the dark band in the M lane in all controls. (C) Lanes 1 and 2 were treated DNAfrom normal lymphocytes. Lanes 3 to 6 were methylation positive control. Lane 7 was DNA from normal lymphocytes and PCRamplified using wild-type primer for monitoring bisulfite treatment. Lane 8 was a negative control (H2O). The results showed that

the F2 fragment of HOXA10 gene from endometriosis patients was partially methylated compared with the controls.

freezer. Menstrual blood was stored in freezers within 30minutes after collection on sanitary napkins and latertransferred to a �80(C freezer. The research wasapproved by the Institutional Review Board of MCW.

Methylation-specific PCR (MSP)

All samples were initially screened by MSP at HOXA10,which is about 3.71 kb, and its structure is schematicallydepicted in Figure 1. Using the MethPrimer software19

(downloadable at http://itsa.ucsf.edu/wurolab/meth-primer), we identified 3 CpG-rich fragments inHOXA10, 1 (F1) in the 50 upstream of exon 1, and 2(F2 and F3) in the intronic region sandwiched by exons1 and 2.

Sixty ng of bisulfite-modified DNA was used for eachMSP assay. The primers designed for F1 and F2 frag-ments were as follows: (1) F1 fragment, methylated:forward 50-GTTT TTATAGTTTTCGGTTTTCGG-30,and reverse 50-ACTCCCAATTTAATTTCGTAAACG-30; unmethylated: forward 50-TTTTTATAGTTT-TTGGTTTTTGG-30, and reverse 50-CACTCCCAATT-TAATTTCATAAACAC-30; (2) F2 fragment, methyl-ated: forward: 50-ATAAGTTTATTAATCGGCGAA-

GTTC-30, and reverse 50-AATAAAAA AAACA AAA-AAAACCGAT-30; unmethylated, forward 50-AAGTT-TATTAATTGGTGAAGTT TGA-30, and reverse50-CCAATAAAAAAAACAAAAAAAACCA-30. Theprimers were designed using the MethPrimer program.19

Genomic DNA extracted from peripheral blood ofhealthy individuals was aliquoted into 2 parts: 1 wastreated with methylase SssI (New England Biolab,Beverly, Mass) as a positive control, and the otherreceived no treatment and was served as a negativecontrol. After methylase treatment, the positive controlwould have 100% methylated cytosines in all cytosinesites.

Bisulfite sequencing of HOXA10 gene

The methylation statuses were further validated bybisulfite sequencing, which is considered to be the goldstandard for methylation evaluation.20 Genomic DNAextracted from endometrial tissue was modified withsodium bisulfite following the manufacturer’s in-structions (Zymo Research, Orange City, Calif). Thismodification resulted in a conversion of unmethylatedcytosine to thymine, whereas the methylated cytosine

374 Wu et al

Figure 3 Bisulfite sequencing of HOXA 10 F3 fragment using bisulfite modified genomic DNA from endometrium ofendometriosis patients (A and B), normal control woman (C), and normal blood lymphocytes (D), and normal genomic DNAtreated with methylase SssI as 100% methylation positive control (E). Methylated CpG sites (red arrows indicated). Unmethylated

CpG sites (black arrows indicated).

remained unchanged.20 A total of 60 ng of bisulfite-modified DNA was subjected to PCR amplification anddirectly sequenced using the ABI 3700 automatedsequencing system (Applied Biosystems, Foster City,Calif). The primers for bisulfite sequencing were designedto complementary to DNA fragments containing noCpG dinucleotides, thus allowing unbiased amplificationof methylated and unmethylated alleles. The followingprimers were designed to amplify CpG rich regionswithin the HOXA10 gene: (1) F1 fragment: forward, 50-GTTGGGGTAGTTTTTATAGTTTT-30, and reverse,50–ATAACCCTTTCTAACTAACATTTCTTATAC-30;(2) F2 fragment: forward 50-GGA GGAT TTAGAGTTTTGGTTTTT-30, and reverse 50-AACCCTTCTC-TAACTCTCAA CTCAA-30; (3) F3 fragment: forward50-TGTGATTTGGGATATTT TTTTTATG-30, and re-verse 50-AAAACTCC TTCTCCAACTCCAATAT-30.

Quantitative real-time RT-PCR

OCT (Sakura Finetek, Torrance, Calif) embedded-frozen endometrium tissue sections were stained usingHistogene LCM Frozen Section Staining solutions(Arcturus, Mountain View, Calif). The glandular epi-thelial cells were dissected by laser capture microdissec-tion. Total RNA was extracted from the capturedepithelial cells using the Picopure RNA isolation kit(Arcturus) and treated with RNase-free DNase I (In-vitrogen, Carlsbad, Calif). The total RNA (10 ng) wasthen reverse-transcribed (Superscript II Reverse Tran-scriptase, Invitrogen), and the HOXA10 mRNAamounts were quantified by a real-time RT-PCR anal-ysis using SYBR Green I detection kit (Qiagen, Valen-cia, Calif). PCR reactions were carried out on the ABIPRISM 7900HT Sequence Detection System (Applied

Wu et al 375

Figure 4 F1 fragment containing 22 CpG sites started from �25 bp to �300 bp 50upstream of exon1. The result demonstratedmethylation levels of endometrium samples from 6 endometriosis patients and 9 healthy control individuals. Gray scale shown atthe left represents the level of methylation for each CpG site.

Biosystems). The amount of HOXA10 mRNA of eachsubject was normalized with that of 18S mRNA(endogenous control, Ambion, Austin, Tex) takenfrom the same subject and the relative amount ofHOXA10 mRNA was calculated using relative quanti-tation of gene expression (Applied Biosystems). The

primers selected for RT-PCR yielded 210 bp PCRproduct from HOXA10 mRNA. The HOXA10mRNA primers were: 50-GCCCTTCCGAGAGCAGCAAAG-30 and 50-AGGTGGACGCTGCGGCTAATC-TCTA-30. The RT-PCR products were checked ona 0.8% agarose gel.

376 Wu et al

Figure 5 Bisufite sequencing of F2 containing 21 CpG sites. The result demonstrated methylation levels of endometrium samplesfrom 6 endometriosis patients and 6 healthy control individuals. Gray scale shown at the right represents the level of methylation for

each CpG site.

Data analysis

The methylation patterns of each subject were listedalong CpG sites sequenced for a given region. Foreach CpG site, no methylation was coded as 0, partialmethylation, 0.5, and complete methylation, 1. Partialmethylation occurred when equal or nearly equalamounts of C and G were present in the sample,manifested as an identical or nearly identical peak inthe SEQUENCER, thus precluding an unambiguousbase calling. For each subject, scores across allsequenced CpG sites in the same region were added.

The difference in scores between the cases andthe controls was compared and evaluated by a ran-domization test by adding scores in a group (case orcontrol). One hundred thousand permutations wereconducted to arrive the empirical P value, p ¼

Pi Ii=n,

where n is the number of permutations, and Ii is theindicator variable indicating whether or not the totalscore Si in the patient group at the ith permutationexceeds the observed score S0. For gene expressiondata, a one-sided t test was used. All computationswere carried out with R (version 1.9.0, www.r-project.org).

Wu et al 377

Figure 6 Bisufite sequencing of F3 fragment containing10 CpG sites. The result demonstrated methylation levels of endometriumsamples from 6 endometriosis patients and 8 healthy control individuals. Gray scale shown at the right represents the level of

methylation for each CpG site.

Results

In MSP assay of 14 endometrial tissue samples (6 cases,3 tubal ligation controls, and 5 menstruum controls) atF2, both methylated and unmethylated bands wereobserved in all 6 endometriosis patients but only 1control. All other 7 controls showed only unmethylatedbands (Figure 2). The MSP assays of F1 fragmentyielded similar results (data not shown). For F3, theMSP assay was not performed.

The MSP results were further validated by bisulfitesequencing. The representative result is shown in Figure3, where samples from endometriosis patients showedboth C and T in all 3 CpG sites. In contrast, only Tswere present in normal controls. The result also in-dicated partial methylations occurred in patients withendometriosis (Figure 3).

The results of the bisulfite sequencing of F1, F2, andF3 fragments were presented schematically in Figures 4,5, and 6. As shown in Figure 4, F1 fragment contains 22

378 Wu et al

Table Real-time RT-PCR quantitative amounts of HOXA10 and 18S mRNA in endometrium samples

HOXA10 18s HOXA10 levelnormalized to 18sSubject ID (phase) Ct mRNA (ng) Ct mRNA (ng)

P1 (P) 31.05 1.2884 20.77 6.9469 0.1855P3 (LP) 29.07 1.5062 19.06 19.0505 0.0791P4 (ES) 26.22 1.8881 18.29 30.0025 0.0629P5 (EP) 31.38 1.2552 20.58 7.7182 0.1626Mean (SE) 1.484 (0.291) 15.93 (7.718) 0.123 (0.061)

C2 (EP) 32.15 1.1799 24.72 0.6693 1.7629C3 (ES) 31.48 1.2443 24.30 0.8595 1.4477Mean (SE) 1.212 (0.046) 0.764 (0.134) 1.605 (0.223)

Ct, Cycle number at which RNA reached a threshold amount of fluorescence above background; ES, early secretory phase; EP, early proliferative phase;

LP, late proliferative phase. P1,3,4,5 = patients with endometriosis; C2 and C3 = controls without endometriosis.

CpG sites, and all samples were successfully sequenced.The randomization test based on 100,000 permutationsresulted in a P value less than .0001, which is highlysignificant. Figure 5 demonstrates the methylationpatterns of the F2 fragment of 12 individuals afterbisulfite sequencing. Three individuals (C2, D1, and D2)were not shown in the figure because of failed PCRamplifications. The pattern was statistically highlysignificant, and extremely unlikely because of chance(P ! .00001, based on 100,000 permutations). For theF3 fragment, 14 samples were successfully sequenced,and the results were shown in Figure 6. The randomi-zation test based on 100,000 permutations resulted ina P value of less than .0001. These results indicate thatHOXA10 was hypermethylated in the 3 fragmentsexamined in the endometrium of women with endome-triosis compared with that of women without.

For 4 women with endometriosis and 2 without, fromwhom total RNAs were available, the expressionof HOXA10 was evaluated by quantitative real-timeRT-PCR (Table). In 2 women without endometriosis,the expression level of HOXA10 gene was significantlyhigher than that of 4 women with endometriosis (P =.0295, one-sided t test test). This agreed with the findingby Taylor et al.17

Comment

As a homeodomain transcription factor, HOXA10 isdynamically expressed in uterine endometrium, where itis necessary for embryo implantation. In human endo-metrium, HOXA10 is expressed in both epithelial andstromal cells, and the expression has been shown to bedriven by sex steroids, with peak expression occurring inthe midsecretory phase,14,15 paralleling with rising pro-gesterone levels during the same stage. In women withendometriosis, the endometrium failed to show theexpected midsecretory rise in HOXA10 gene expression,and its cause remains unclear.17 Most women with

endometriosis show normal sex steroids production,and it has been shown that the estrogen receptor (ER)and progesterone receptor (PR) activities in the eutopicendometrium of women with endometriosis were nodifferent from those of women without the disease,21

although in the latter the results appeared to bemixed.22,23 Hence, a question naturally arises as towhat causes the apparent down-regulation of HOXA10in the endometrium of women with endometriosis. Itshould be noted that while both ER and PR activities inepithelium diminish sharply in mid and late secretoryphases, their activities in stroma are somewhat pre-served.24-27 In addition, the expression of aromataseP450, which synthesizes estrogen, has been reported tobe increased in the eutopic endometrium of women withendometriosis.28 Because sex steroids induce HOXA10expression in the endometrium, its aberrant expressionmay be attributable to either aberrant local productionof steroids and altered, perhaps cell-specific, ER and/orPR activity level in the endometrium. These observationssuggest a possibly complex mechanism in regulatingHOXA10 gene expression in both epithelium andstroma, for example, PR or ER induction of HOXA10expression in epithelium as a paracrine event mediated bythe stroma.29 Even if this is indeed the case, one questionstill lingers: what, then, causes the changes in either localproduction of sex steroids or the activities of theirreceptors? Because most endometriosis, once diagnosed,does not go away without any intervention, there must besome mechanism that maintains these aberrations thatresult in aberrant expression of HOXA10.

This paper suggests an entirely different and biolog-ically plausible mechanism for the observed down-regulation of HOXA10, that is, aberrant methylationmay be responsible for aberrant gene expression. As ithas become abundantly clear, methylation of promoterregions is associated with a compacted chromatinstructure, accompanying transcriptional silencing ofthe affiliated gene.9,30,31 Methylation-induced epigeneticchanges play an important role in development,32 and

Wu et al 379

can also occur with age.33 Because methylation changesare heritable in cell divisions, epigenetic modification ofthe genome through methylation provides a mechanismfor maintaining stable gene activity states. In addition,because methylation states are reversible, they can bemodified by environmental or lifestyle factors which, inturn, may contribute to the development of abnormalphenotypes.10

Several cautionary words are in order. First, becausethis study is a retrospective one, the demonstratedaberrant hypermethylation in the 50 upstream and thefirst intron of HOXA10 gene and the reported (andconfirmed in our own study) aberrant expression ofHOXA10 in the endometrium of women with endome-triosis may, of course, be merely an association.However, given that hypermethylation in the promoterregions tend to suppress the gene expression, and thatmethylation provides a mechanism for maintainingstable gene activity states, there is reason to believethat the aberrant expression at HOXA10 in the endo-metrium of women with endometriosis may result fromaberrant methylation. In addition, that both HOXA10and methylation are involved in development, thatmethylation changes with age, and that age is the onlysociodemographic risk factor consistently reported forendometriosis,34 all these observations appear to suggestthat it is not only biologically plausible but also highlylikely that aberrant methylation at HOXA10 may beresponsible for its aberrant expression. Last, our studyhas been confined to patients with stages III-IV endo-metriosis. Whether our results hold up for early stagesof endometriosis await for further studies.

In this paper, we did not evaluate methylation patternsin matched endometriotic tissues because the interest inHOXA10 has been mainly in its role in endometrialdevelopment and in implantation.12 So far, there has beenno report on its expression levels in ectopic endometrium.Because our main focus was to provide a biologicalplausible explanation to possible mechanisms underlyingaberrant expression of HOXA10 in the endometrium ofwomen with endometriosis, naturally we only evaluatedeutopic endometrium. HOXA10 gene may or may nothave aberrant expressions or even aberrant methylation,as with many other genes. Even if there are aberrantexpressions or aberrant methylations, it is possible thatthese changes may be a consequence of endometriosis.There are indications in the literature that genetic orepigenetic alterations are found in histologically normaltissue adjacent to primary tumor.35

Epigenetic effects by means of DNA methylationhave been demonstrated to have important roles indevelopment, tumorigenesis, and other diseases.9 To ourknowledge, this is the first report that the HOXA10 genein the endometrium from women with endometriosis hasaberrant hypermethylations, and suggests that endome-triosis, like neoplasia, may also be an epigenetic disease.

By and in itself, this aberrant methylation is worthfurther investigation. Although Martini et al36 examinedmethylation changes at hMLH1 and p16 in women withendometriosis, their purpose was mainly to see whetherthe aberrant methylation at these genes could be used topredict malignant transformation because of involve-ment of these genes in neoplasia.

We also note that menstrual blood can be a valuable,abundant, and noninvasive source for detection ofmethylation changes, as has been very recently reportedalso by Fiegl et al.37 If aberrant methylation atHOXA10 or other genes in endometrium turn out tobe the cause for their aberrant expressions, which, inturn, are responsible for abnormal uterine receptivityand implantation capability, menstrual blood can,perhaps, be used for screening and diagnosis.

The partial methylation at HOXA10 observed inour study could be caused by cell type heterogeneity(stroma, epithelium, etc), or genuine methylation het-erogeneity, or both. One way to resolve this is byselective examination of different cell types through,say, laser capture microdissection (LCM). However,harvesting sufficient cells by LCM enough for bisulfitesequencing without introducing further heterogeneitycould pose a big challenge that needs to be resolved infuture studies. In addition, a more definite proof thataberrant methylation at HOXA10 is the cause for itsaberrant expression in the endometrium of women withendometriosis also awaits future studies.

In summary, we have demonstrated that there areaberrant methylations at HOXA10 that may be re-sponsible for its aberrant expression in the endometriumof women with endometriosis. This suggests a possibilitythat endometriosis may be also an epigenetic disease. Ifthis is indeed the case, this perspective should open upnew ways to the delineation of endometriosis pathogen-esis, and might also lead to novel ways to treat thedisease, for example, reversing aberrant methylationthrough pharmacologic means.38

Acknowledgments

We thank Vicki Magnuson, Kerry Nolan, RachelKraemer, and Nancy Rotzel for their excellent technicalassistant of bisulfite sequencing, and Michael Jensen-Seaman for providing important Genome databaseinformation. We also thank volunteers for donatingtheir materials that made this study possible.

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