Original Article
Aberrant HOXA10 Methylation in Patients
With Common Gynecologic Disorders:
Implications for Reproductive Outcomes
Jennifer L. Kulp, MD
1
, Ramanaiah Mamillapalli, PhD
1
,
and Hugh S. Taylor, MD
1
Abstract
HomeoboxA10 (HOXA10) is a transcription factor that is crucial for the development and patterning of the uterus during
embryogenesis. In the adult endometrium, HOXA10 expression is regulated by steroid hormones and embryonic signals.
Expression of sufficient HOXA10 messenger RNA is essential to endometrial receptivity and embryo implantation. Aberrant
methylation is believed to alter the expression of HOXA10. Methylation of this gene may be associated with decreased fertility,
implantation defects, and/or reproductive wastage seen in certain disease states that affect the female reproductive tract. This
study describes the differences in methylation patterns of HOXA10 gene in uterine myomas, endometriosis, uterine septum,
Asherman syndrome, or uterine polyps of women undergoing hysteroscopic surgery. In the endometrium of uteri with polyps,
submucosal myomas, and intramural myomas, there were CpG sites within the HOXA10 gene that were highly methylated
compared to controls. The HOXA10 gene in women with endometriosis was hypomethylated compared to controls. DNA
methylation may be a common molecular mechanism that results in reproductive dysfunction seen in gynecologic disease.
Keywords
HOXA10, DNA methylation, endometriosis, endometrium
Introduction
Many common gynecologic conditions such as endometriosis
or uterine myomas are associated with infertility.
1-3
Embryo
implantation defects are at least partly responsible for the
decreased fertility seen in women with these conditions.
2,4-8
The HOX/Hox (human/rodent) family of genes encode tran-
scription factors that have a role in embryo development and
particularly in female reproductive tract.
9-11
HomeoboxA10
(HOXA10) is expressed in the uterus of the developing
embryo.
12
In adult humans, HOXA10 is expressed in endome-
trial glands and stroma. Its expression peaks during the mid-
luteal phase of the cycle, which coincides with the time of
endometrial receptivity to embryo implantation.
13-15
Expres-
sion of sufficient HOXA10 is known to be essential to endo-
metrial receptivity and embryo implantation.
16-18
This is
clearly demonstrated in the HOXA10 knockout mouse, which
is sterile secondary to impaired endometrial receptivity. The
HOXA10
/
mouse produces viable embryos, yet neither
HOXA10
/
nor wild-type embryos implant. However, when
the HOXA10
/
embryos are transfe rred to the wild-type
mouse, they implant and develop normally.
19-21
Likewise, in
humans, cyclic expression of HOXA10 in the adult endome-
trium
9,13
is required for endometrial receptivity. In several
common gynecologic disease states that are associated with
implantation defects, such as endometriosis, hydrosalpinges,
polyps, and submucosal myomas, defective HOXA10 expres-
sion has been demonstrated.
22-29
DNA methylation regulates gene expression
30-32
and has
been associated with several inflammatory diseases and can-
cers.
33-36
Aberrant methylation is known to alter the expression
of HOXA10.
37
Methylation of this gene may be associated with
decreased fertility, implantation defects, and/or reproducti ve
wastage seen in certain disea se sta tes tha t affec t the female
reproductive tract.
38-41
Our objective was to examine methy-
lation of the HOXA10 gene in t he endometrium in disease
states that affect the female reproductive tract and embryo
implantation, specifically uterine myoma, endometriosis,
uterine septum, hydro salpinges, uterine polyps, and Asher-
man syndrome. We hypothesized that methylation of the
HOXA10 gene may be a common mechanism explaining the
1
Department of Obstetrics, Gynecology and Reproductive Sciences, Yale
School of Medicine, New Haven, CT, USA
Corresponding Author:
Ramanaiah Mamillapalli, Department of Obstetrics, Gynecology and Repro-
ductive Sciences, Yale School of Medicine, 333 Cedar Street, PO Box 208063,
New Haven, CT 06510, USA.
Email: ramana.mamillapalli@yale.edu
Reproductive Sciences
2016, Vol. 23(4) 455-463
ª The Author(s) 2016
Reprints and permission:
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DOI: 10.1177/1933719116630427
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aberrant ex pre ssio n of the gene a n d repr oduc tiv e dys func tion
observed in these disorders.
Materials and Methods
Sample Collection
We collected endometrial samples from a total 84 women with
submucosal uterine myomas (n ¼ 13), intramural uterine myo-
mas (n ¼ 13), endometriosis (n ¼ 27), uterine septum (n ¼ 6),
Asherman syndrome (n ¼ 8), hydrosalpinx (n ¼ 4), or uterine
polyps (n ¼ 11) undergoing hysteroscopic surgery as well as
endometrial samples from controls (n ¼ 7) who were anon-
ymous egg donors free from gynecologic disorders undergoing
oocyte retrieval. Some women had more than 1 condition (ie,
endometriosis and hydrosalpinx) and were included separately
in each group (Table 1). This study was approved by the Yale
University Human Investigation Committee. At the time of
biopsy, each endometrial tissue sample was stored in 1 mL of
RNAlater (Qiagen, Valencia, California) at 80
C until RNA
and DNA isolation.
Quantitative Real-Time Polymerase Chain
Reaction Analysis
The RNA was extracted using the RNeasy Mini kit (Qiagen),
according to the manufacturer’s protocol. Messenger RNA
(mRNA) levels were analyzed by quantitative real-time reverse
transcriptase-polymerase chain reaction (qRT-PCR) using the
Bio-Rad iCycler iQ system (Bio-Rad Laboratories, Hercules,
California). For each sample, 500 ng of total RNA was reverse
transcribed using an iScript cDNA Synthesis Kit (Bio-Rad
Laboratories). Real-time RT-PCR was performed using a
MyiQ Single-Color Real-Time PCR Detection System and iQ
SYBR Green Supermix (both from Bio-Rad Laboratories). The
sequences of all primers and the real-time RT-PCR reaction
conditions have been described previously.
42
Each assay was
run in duplicate with each set of primers, and samples without
mRNA were included as negative controls. HomeoboxA10
gene expression was normalized to the expression of b-actin
for each run. Relative mRNA expression for each gene was
calculated using the comparative cycle threshold (Ct) method
(known as 2
DDCT
method).
43-44
Results are presented as the
mean + standard error (SE). Statistical significance was deter-
mined and analyzed by Student t test. P values less than .05
were considered statistically significant.
DNA Isolation and Methylation
DNA was isolated using t he DNeasy Mini Kit (Qiagen),
according to the manufacturer’s protocol. Quantitative DNA
methylation analysis was performed on the collected tissue,
using Sequenom MassARRAY quantitative methylation anal-
ysis and EpiTyper technology, in 3 CpG-rich fragments in 2
regions of the HOXA10 gene (total 39 distinct CpG sites/
fragments), to detect differences in methylation. The first CpG
island is located 50 base pair upstream of exon 1, and 2 addi-
tional islands are located in the intron flanked by exons 1 and 2.
Briefly, genomic DNA underwent bisulfite treatment followed
by PCR amplification using a T7-promotor tag. In vitro RNA
transcription was performed followed by uracil-specific clea-
vage. Matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry was used to analyze the cleavage products
and produced distinct signal pattern pairs indicating nonmethy-
lated and methylated DNA. Due to limitations of this tech-
nique, there were a few cleavage products that were
indistinguishable as they have the same mass. In fragment 1,
sites CpG37 and CpG4, sites CpG38 and CpG21, and sites
CpG17 and CpG30 could not be distinguished from each other.
Similarly, sites CpG8 and CpG9 in fragment 2 were indistin-
guishable. Differences in endometrial HOXA10 expression and
mean percentage methylation at different CpG areas between
the disease groups and controls were determined using Student
t test. P < .05 was considered statistically significant. To deter-
mine whether there was an association between the level of
methylation and gene expression, a Pearson product-moment
correlation coefficient was calculated.
Results
The CpG islands present in HOXA10 gene are shown in
Figure 1. The CpG sites or clusters present at different regions
in HOXA10 gene are designated as fragment 1 if in the pro-
moter region, while fragments 2 and 3 are located in the intron
regions. The summary of the Sequenom MassARRAY quanti-
tative methylation analysis of the CPG areas located in the 5
0
promoter region (fragment 1) is shown in Figure 2, while in the
intron region (fragment 2 and 3) is shown in Figures 3 and 4.
The average percentage methylation value at each numbered
CpG site or cluster of CpG sites for the 84 endometrial samples
is shown. The results show the overall low levels of methyla-
tion. There were very few CpG sites with greater than 50 %
methylation. However, some individual sites were highly and
specifically methylated. The number of CpG sites with
Table 1. Patients’ Characteristics.
a
Condition
Age
(Mean + SD),
years
Gravity
(Mean)
Parity
(Mean)
Controls (n ¼ 7) 34.9 + 3.9 1.7 1.1
Asherman (n ¼ 8) 33.5 + 3.3 1.4 0.5
Hydrosalpinx (n ¼ 4) 37.5 + 5.8 1.5 1
Endometriosis (n ¼ 27) 32.3 + 6.7 1.1 0.7
Intramural myoma (n ¼ 13) 39.2 + 7.6 1.5 1
Submucosal myoma (n ¼ 13) 36.6 + 6.2 0.6 0.6
Polyp (n ¼ 11) 33.8 + 3.7 0.5 0.5
Septum (n ¼ 6) 32.7 + 5.1 1.2 1.2
Abbreviation: SD, standard deviation.
a
N sums to greater than 84 as there was an overlap of patients’ diagnosis.
There were 6 patients with more than 1 diagnosis: 2 with submucosal myomas
and septum, 1 with history of endometriosis and polyp, 1 with endometriosis
and hydrosalpinx, and 1 with endometriosis and submucosal myoma.
456 Reproductive Sciences 23(4)
significantly altered methylation status compared to controls
by disease is shown in Table 2. Specifically, CpG17 and CpG4
in the promoter region and CpG12 in the second intronic region
of HOXA10 were highly methylated in the control samples and
had significantly greater percentage methylation than many of
the disease states. At other CpG sites, the disease state endo-
metrium had CpG sites with significantly greater percentage
methylation compared to controls. Specifically, in polyp endo-
metrium, 4 of the 6 statistically different CpG clusters in the
promoter region had higher percentage methylation compared
to controls (Figure 2; CpG18-CpG20, CpG21, CpG26, and
CpG27-28). In endometrium of uteri with intramural myomas,
2 of the 4 statistically different CpG clusters in the promoter
region (Figure 2: CpG 21 and 22-25), as well as 1 CpG site in
the second intronic region (Figure 4: CpG13- C pG15), had
higher methylation compared to controls. In endometrium of
uteri with submucosal myomas, 3 of the 4 statistically different
CpG clusters in the promoter region had highe r degrees of
methylation compared to controls. Interestingly, endometrium
of submucous and intramural myomas as well as polyp had two
of the s ame CpG sites or clusters, CpG21 and CpG cluster
(CpG_22.23.24.25) located in the promoter region, hyper-
methylated compared to controls. Two of the diseases (submu-
cosal myoma and polyp) shared a second CpG cluster
(CpG_12.13.14.15.16) also located in the promoter, which had
greater methylation as compared to controls in both. Submu-
cous and intramural leiomyoma also showed distinct methyla-
tion differences as well. Overall, the percentage of CpG sites or
clusters within each fragment with statistically significant dif-
ferent methylat ion levels between diseases and controls was
low. However, all diseases studied had at least 1 CpG cluster
in the promoter region of HOXA10, which was differently
methylated when compared to controls. Many of the diseases
also demonstrated at least 1 CpG cluster with methylation sig-
nificantly different from controls in intronic fragment 2 and
few diseases were associated with differences in methylation
in intronic fragment 3 as shown in Figures 1, 3, and 4.
When percentage methylation was averaged for each CpG
island or fragment in disease states and compared to controls, 3
diseases (submucosal myoma, polyp, and endometriosis) were
found to have significantly different CpG island wide percent-
age methylation compared to controls. In submucosal myoma,
fragment 2 had 10% methylation compared to 14% in controls
(P ¼ .008) and fragment 3 had 12% methylation compared to
16% in controls (P ¼ .0 14). In polyp, fragment 3 had 12%
methylation compared to 16% in controls (P ¼ .049). In endo-
metriosis, all fragments were significantly less methylat ed
compared to controls—fragment 1: 16% versus 21% (P ¼
.004); fragment 2: 8% versus 14% (P ¼ .002); fragment 3:
13% versus 16% (P ¼ .006). In all cases, the disease states
were found to have less overall methylation compared to con-
trols. Percentage methylation was also averaged over the whole
HOXA10 gene. Gene-wide methylation was found to be sig-
nificantly different only in 1 disease, endometriosis, compared
to controls (14% vs 17%, P ¼ .001).
Quantitative real-time reverse transcriptase-polymerase
chain reaction was performed to assess the expression levels
of HOXA10 in disease and normal endometrium. RNA was
available from 43 disease state endometrial samples. The
RT-PCR results for the disease samples were normalized to
the controls, and these data were correlated with percentage
DNA methylation at significantly different CpG sites or clus-
ters. There was a correlation between DNA methylation and
gene expressio n in endo metriu m from sub muc ous myo ma at 1
CpG cluster in the second intronic region (Figure 4; CpG
4.5.6; r ¼ .72, P ¼ .02). There was also a correlation in
endometriosis between CpG site 11 methylation in the second
intronic region and gene expression (r ¼.9, P ¼ .0 4) . We
also observed that fragment-wide DNA methylation for frag-
ment 3 in endometriosis was also correlated with gene expres-
sion ( r ¼.9, P ¼ .04).
Discussion
Our study reports the differential methylation of HOXA10 CpG
sites in human female reproductive diseases such as submuco-
sal myoma, endometriosis, intramural myoma, septum, polyps,
hydrosalpinx, and Asherman syndrome compared to normal
female samples. These data suggest that the HOXA10 gene
may be differently methylated in multiple diseases and that
DNA methylation may be a common means by which these
gynecologic diseases affect the HOXA10 gene and its expres-
sion in female reproductive track. DNA methylation has not
been well studied in most of the gynecological diseases
Figure 1. HomeoboxA10 (HOXA10) gene structure. The genomic region of HOXA10 showing the locations of the CpG islands. The CpG sites
or clusters present on the gene are designated as fragments 1, 2, and 3. Intron region containing 2 clusters of CpG islands (fragment 1 and 2),
while promoter region having 1 cluster (fragment 1).
Kulp et al 457
mentioned previously, except for endometriosis and uterine
polyps.
45-48
Overall, we noted low levels of methylation of the
HOXA10 gene in both disease and control endometrium. A few
highly specific sites had high levels of methylation in controls.
Methylation is associated with the absence of disease at these
sites, and loss of methylation can signal disease. The net
absence or presence of methylation is less important than the
specific pattern of methylation measured at the level of indi-
vidual C/G base pairs.
All diseases had at least 1 CpG site that was significantly
differently methylated as compared to normal. Yet each disease
had a unique pattern of methylation of the HOXA10 gene, and
Figure 2. Summation of Sequenom MassARRAY methylation analysis of HomeoboxA10 (HOXA10) CpG islands in the promoter region in each
disease state. The percentage DNA methylation of individual CpG sites or clusters, averaged for each disease state, from controls is indicated by the
bars (blue is disease and red is controls). Each graph represents a different disease, submucosal myoma (A), endometriosis (B), intramural myoma (C),
septum (D), polyp (E), hydrosalpinx (F), and Asherman syndrome (G). The CpG positions from each genomic region analyzed are numbered in
ascending order. Some CpG sites are grouped because of the limits of the technique, which do not allow them to be resolved individually. *Particular
CpG sites or clusters with significantly different methylation levels compared to controls. An absent bar indicates that insufficient methylation data
were obtained due to very low levels of methylation. (The color version of this figure is available in the online version at http://rs.sagepub.com/)
458 Reproductive Sciences 23(4)
methylation of some CpG sites appears to be disease specific.
This suggests that there may be disease-specific mechanisms of
HOXA10 methylation and gene regulation. Further, aberrant
methylation of the HOXA10 gene is likely related to its aber-
rant expression, and this aberrant expression is thought to have
a role in the reproductive dysfunction such as implantation
failure seen in these diseases, for example, in endometriosis
as reported previously.
37
CpG sites located in the promoter region of HOXA10 had
significantly increased methylation compared to normal in 3
diseases: polyp, intramural myoma, and submucosal myoma.
CpG21 was consistently methylated. CpG methylation,
Figure 3. Summation of Sequenom MassARRAY methylation analysis of HomeoboxA10 (HOXA10) CPG island intronic region 1 (fragment 2) in each
disease state, submucosal myoma (A), endometriosis (B), intramural myoma (C), septum (D), polyp (E), hydrosalpinx (F), and Asherman syndrome (G).
*Particular CpG sites or clusters with significantly different methylation levels compared to controls. Percentage DNA methylation is representedbythe
bars, with disease state showed in blue and controls in red. (The color version of this figure is available in the online version at http://rs.sagepub.com/)
Kulp et al 459