Abstract

BACKGROUND

Epigenetic regulation may be involved in modulation of gene expression during the normal cyclic changes of the human endometrium. We investigated expression of DNA methyltransferases (DNMTs) in endometrium during the menstrual cycle and the influence of sex steroid hormones on DNMT in endometrial stromal cells (ESC) in culture.

METHODS

Expression of DNMT1, DNMT3a and DNMT3b was assessed by immunohistochemistry and real-time RT–PCR in endometrial tissue (n = 42 women). ESC (n = 3 women) were cultured with estradiol and medroxyprogesterone acetate (E + MPA) for 17 days, and DNMT mRNA levels were measured by real-time RT–PCR.

RESULTS

Nuclei of both epithelial and stromal cells immunostained for DNMT1, DNMT3a and DNMT3b during each phase of the menstrual cycle. Tissue levels of DNMT1 and DNMT3a mRNA were significantly lower in the mid-secretory phase than in the proliferative phase (P < 0.01). For DNMT3b, the change in mRNA levels showed a similar trend to that for DNMT3a. In ESC culture, DNMT3a and DNMT3b mRNA levels were significantly decreased by E + MPA treatment (P < 0.01 and P < 0.05, respectively) at Day 8 and Day 17.

CONCLUSIONS

DNMT mRNAs declined in the human endometrium during the secretory phase, and E + MPA down-regulated DNMT3a and DNMT3b mRNAs in ESC in culture. These results suggest that DNMTs have regulatory functions in gene expression that is associated with decidualization.

Introduction

The human endometrium, which mainly consists of endometrial epithelial cells and endometrial stromal cells (ESC), has cyclic changes in morphology and in function depending on female sex steroid hormone exposure. These cells actively proliferate under estrogen exposure during the proliferative phase and thereafter differentiate under progesterone exposure during the secretory phase. A number of genes are involved in proliferation, differentiation and tissue breakdown in the endometrium under the influence of female sex steroid hormones (Sugino et al., 2002a, 2004; Ace and Okulicz, 2004; Ponnampalam et al., 2004). A great number of genes are up-regulated or down-regulated in the human endometrium during decidualization, which occurs around the time of embryo implantation (Popovici et al., 2000; Kao et al., 2002; Okada et al., 2003; Riesewijk et al., 2003; Ace and Okulicz, 2004; Mirkin et al., 2005), suggesting the presence of complex mechanisms of gene expression. However, little is known about the molecular mechanisms involved in the regulation of gene expression in the human endometrium.

In the last decades, it has become clear that epigenetic regulation, including DNA methylation and histone modification, plays a key role in transcriptional regulation. DNA methylation occurs at cytosines within CpG dinucleotides that are clustered frequently in regions of ∼1–2 kb in length, called CpG islands, in or near the promoter and first exon regions of genes (Esteller et al., 2002; Jones and Baylin, 2002). DNA methylation at the CpG dinucleotides is a post-replication event catalyzed by DNA methyltransferase (DNMT) (Smith, 1994) that adds a methyl group to the cytosine ring to form methyl cytosine, which establishes normal methylation patterns during embryogenesis and reproduces these patterns during replication of adult cells (Li et al., 1993; Razin and Kafri, 1994). DNA methylation is an important mechanism of epigenetic gene regulation, and is involved in genomic imprinting, X chromosomal inactivation, aging, mutagenesis and regulation of tissue-specific gene expression during development and adult life (Li et al., 1993; Razin and Kafri, 1994; Ohgane et al., 1998; Imamura et al., 2001; Li, 2002; Shiota and Yanagimachi, 2002). Aberrant methylation of CpG islands, located in the 5′-promoter region of genes, is commonly associated with transcriptional inactivation (Nan et al., 1998). Such inactivation is well known in various human cancers, especially in tumor suppressor genes (Ushijima and Okochi-Takada, 2005).

Several DNMTs exist. DNMT1 functions as a ‘maintenance’ DNMT in mammalian cells and is therefore responsible for accurately replicating genomic DNA methylation patterns during cell division (Liu et al., 1998). On the other hand, DNMT3a and DNMT3b are thought to catalyze de novo methylation of DNA (Hsieh, 1999). Recent research also shows that DNMT1, DNMT3a and DNMT3b co-operatively maintain DNA methylation (Ting et al., 2004).

Recently, aberrant expression of DNMTs was observed in endometriosis, which is a non-cancerous ectopic growth of the human endometrium (Wu et al., 2007). Aberrant DNA methylation of the promoter region is involved in aberrant gene expression of steroidogenic factor-1 and estrogen receptor in endometriosis (Xue et al., 2007a, b; Utsunomiya et al., 2008). Furthermore, in the eutopic endometrium of women with endometriosis, reduced expression of HOXA10, which is a transcription factor and plays an important role in uterine receptivity, was found to be due to DNA methylation of the promoter region (Wu et al., 2005). In addition, aromatase expression in ESC is under epigenetic regulation (Izawa et al., 2008). Histone acetylation is involved in differentiation of ESC and endometrial epithelial cells (Sakai et al., 2003; Uchida et al., 2005). These reports led us to hypothesize that epigenetic regulation is involved in the normal cyclic changes of the human endometrium. To test this hypothesis, we investigated changes in the expression of DNMTs in the normal endometrium during the menstrual cycle and the influence of female sex steroid hormones on DNMT expression in ESC.

Materials and Methods

This study was reviewed and approved by the Institutional Review Board of Yamaguchi University Graduate School of Medicine. Informed consent was obtained from the women before collection of any samples for this study.

Tissue samples

Endometrial tissues were collected from hysterectomy specimens or biopsies for histological dating of the endometrium in 42 women with regular menstrual cycles (aged 22–50 years, median 36.9 years). All of the women received no steroid medications. Endometria were dated according to the histological criteria by Noyes et al. (1950) and were classified into four different groups: proliferative phase (days 6–14, n = 14), early secretory phase (days 15–18, n = 10), mid-secretory phase (days 19–23, n = 11) and late secretory phase (days 24–28, n = 7). Endometrial samples were snap-frozen in liquid nitrogen and stored at −80°C until RNA isolation. For immunohistochemistry, the tissue specimens were fixed in 10% buffered formalin and embedded in paraffin.

Immunohistochemistry

Immunohistochemistry for DNMTs in the endometrium was performed on 4 µm thick paraffin sections mounted on silane-coated glass slides (Dako, Glostrup, Denmark) using anti-DNMT1 monoclonal antibody (IMG-261 mouse; Imgenex, San Diego, CA, USA), anti-DNMT3a polyclonal antibody (RB1852 rabbit; Abgent, San Diego, CA, USA) or anti-DNMT3b polyclonal antibody (RB1906 rabbit; Imgenex), as reported previously (Sugino et al., 1996, 2002b). Briefly, the sections were deparaffinized in xylene and dehydrated through a graded series of ethanol. For antigen retrieval, the sections were autoclaved at 121°C for 15 min. Endogenous peroxidase activities and non-specific binding were then blocked with 1% H2O2 and 10% normal rabbit serum (Nichirei, Tokyo, Japan) for DNMT1 or 10% normal goat serum (Nichirei) for DNMT3a and DNMT3b, respectively. The sections were then incubated with the primary antibody diluted 1:50 overnight at 4°C. Parallel control sections were incubated with normal mouse serum or normal rabbit serum (Dako) instead of specific primary antibodies. Biotinylated antimouse antibody (Nichirei) for DNMT1 or biotinylated antirabbit antibody (Nichirei) for DNMT3a and DNMT3b was used as the secondary antibody. After the sections were rinsed in phosphate-buffered saline (PBS), they were incubated in streptavidin–peroxidase complex (Nichirei) for 5 min, rinsed in PBS and then visualized with diaminobenzidine and counterstained with hematoxylin. Dark brown nuclear staining indicated a positive reaction. The histological sections were independently evaluated by three observers, and relative intensities of the signals were estimated at + (weakly positive) to +++ (strongly positive).

ESC culture

For ESC culture, endometrial tissues that were histologically diagnosed as being in the late proliferative phase were used. Tissue samples were obtained from three individuals, and cells from an individual were cultured in triplicate. ESC were isolated as reported previously (Sugino et al., 2000, 2002c). Endometrial tissues were washed with phenol red-free Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Paisley, UK) containing 4 mmol/l glutamine (Invitrogen), 50 µg/ml streptomycin (Invitrogen) and 50 IU/ml penicillin (Invitrogen), and minced into small pieces of <1 mm3. After the enzymatic digestion of minced tissues with 0.2% collagenase (Sigma, St Louis, MO, USA) in a shaking water bath for 2 h at 37°C, ESC were separated by filtration through a 70 µm nylon mesh. The filtrates were washed three times, and the number of viable cells was counted by trypan blue dye exclusion. The homogeneity of the stromal cell preparation (98%) was verified by immunocytochemistry using an antibody against vimentin, a specific marker of stromal cells. ESC were seeded at 105 cells/cm2 in 75 cm2 tissue culture flasks and grown until confluence in phenol red-free DMEM containing glutamine, antibiotics and 10% dextran-coated charcoal-stripped fetal calf serum (FCS) (Biological Industries, Kibbutz Beit Haemek, Israel) at 37°C, 95% air and 5% CO2. If necessary, cells were subcultured in 75 cm2 tissue culture flasks after the first passage until confluence. For treatments, cells were subcultured into 25 cm2 tissue culture flasks (second or third passage), and the cell culture medium was changed to the treatment medium at 80% confluence.

To examine the effect of estrogen and progesterone on DNMTs mRNA levels in ESC, cells were incubated with treatment medium (phenol red-free DMEM supplemented with glutamine, antibiotics and 2% stripped FCS) containing a combination of estradiol (10−8 M) (Sigma) and medroxyprogesterone acetate (MPA, 10−6 M) (Sigma) for 17 days at 37°C, in 95% air and 5% CO2. The concentrations of estradiol and MPA, and the period of incubation were based on our previous reports (Sugino et al., 2000, 2002d). The medium was changed every other day. Decidualization was confirmed by morphology and mRNA expression of insulin-like growth factor-binding protein-1 (IGFBP-1), which is a specific marker of decidualization (Giudice et al., 1992; Sugino et al., 2000). Total RNA was isolated from cultured cells, and RT–PCR for DNMTs was performed as described below, with a duplicate PCR for each culture.

Real-time RT–PCR

Total RNA was extracted from endometrial tissues and cultured cells using Isogen (Wako, Osaka, Japan), and real-time RT–PCR was performed as reported previously (Asada et al., 2008). RT reactions were performed with ExScript RT reagent kit (TAKARA, Kyoto, Japan) according to the manufacturer's protocol. Briefly, 2 µg of total RNA was incubated with 4 µl of 5× ExCript buffer, 1 µl of dNTP mixture (10 mM each), 1 µl of Random primers (50 µM), 0.5 µl of ExCript reverse transcriptase (200 U/µl) and 0.5 µl of RNase inhibitor (40 U/µl) in 20 µl of reaction mixture at 42°C for 15 min, after which the reverse transcriptase was inactivated by heating the samples at 95°C for 2 min. The complementary DNA (cDNA) was immediately used for PCR. All PCRs were performed using SYBR Premix Ex Taq (TAKARA) and a LightCycler (Roche Applied Science, Basel, Switzerland). Briefly, 2 µl of aliquots containing cDNA were amplified in a total volume of 20 µl containing 4 µl of a 5× SYBR PreMix Ex Taq and 0.2 µM each primer. For internal controls, TATA box-binding protein (TBP) cDNA or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also amplified. According to the previous report (Girault et al., 2003), the following primers were used: DNMT1 (forward, 5′-TACCTGGACGACCCTGACCTC-3′, reverse, 5′-CGTTGGCATCAAAGATGGACA-3′) (product size: 103 bp); DNMT3a (forward, 5′-TATTGATGAGCGCACAAGAGAGC-3′, reverse, 5′-GGGTGTTCCAGGGTAACATTGAG-3′) (111 bp); DNMT3b (forward, 5′-GGCAAGTTCTCCGAGGTCTCTG-3′, reverse, 5′-TGGTACATGGCTTTTCGATAGGA-3′) (113 bp); TBP (forward, 5′-TGCACAGGAGCCAAGAGTGAA-3′, reverse, 5′-CACATCACAGCTCCCCACCA-3′) (132 bp); IGFBP-1 (forward, 5′-CGAAGGCTCTCCATGTCACCA-3′, reverse, 5′-TGTCTCCTGTGCCTTGGCTAAAC-3′) (98 bp) and GAPDH (forward, 5′-AGGTGAAGGTCGGAGTCA-3′, reverse, 5′-GGTCATTGATGGCAACAA-3′) (99 bp). All samples were run in duplicate. For appropriate negative controls, the RNA template was replaced with nuclease-free water in each run. Melting curves of the products were obtained after cycling by a stepwise increase of temperature from 55 to 95°C. At the end of 40 cycles, reaction products were separated electrophoretically on an agarose gel and stained with ethidium bromide for visual confirmation of the PCR products.

Statistical analyses

Statistical analysis was carried out using the Statistical Package for the Social Sciences for windows 13.0. To evaluate whether tissue mRNA levels significantly vary during the menstrual cycle, the Tukey honest significant difference test was used. For ESC cultures, differences in mRNA levels were determined using Duncan's new multiple range test. P < 0.05 was considered to be significant.

Results

Nuclei of both epithelial cells and stromal cells in tissue sections immunostained for DNMT1, DNMT3a and DNMT3b during each phase of the menstrual cycle. Representative results from the late proliferative phase are shown in Fig. 1. The staining intensities did not vary among the menstrual phases.

Figure 1

Immunohistochemical staining for DNMT1, DNMT3a and DNMT3b in the human endometrium. Immunohistochemical staining was performed on tissue samples from the proliferative phase, early secretory phase, mid-secretory phase and late secretory phase. Tissue samples were taken from three different patients in each phase. Representative results from the late proliferative phase are shown. Nuclei of both epithelial cells and stromal cells immunostained for DNMT1 (A), DNMT3a (B) and DNMT3b (C). No immunoreactivity was observed in the control sections incubated with normal mouse serum (D) and normal rabbit serum (E). Bar = 50 µm.

Figure 1

Immunohistochemical staining for DNMT1, DNMT3a and DNMT3b in the human endometrium. Immunohistochemical staining was performed on tissue samples from the proliferative phase, early secretory phase, mid-secretory phase and late secretory phase. Tissue samples were taken from three different patients in each phase. Representative results from the late proliferative phase are shown. Nuclei of both epithelial cells and stromal cells immunostained for DNMT1 (A), DNMT3a (B) and DNMT3b (C). No immunoreactivity was observed in the control sections incubated with normal mouse serum (D) and normal rabbit serum (E). Bar = 50 µm.

Changes in DNMT1, DNMT3a and DNMT3b mRNA levels in the endometrial tissue are shown in Fig. 2. DNMT1 mRNA levels were significantly lower in the mid-secretory phase than in the other menstrual phases (Fig. 2A). DNMT3a mRNA levels were significantly lower in the secretory phase than in the proliferative phase (Fig. 2B), being lowest in the mid-secretory phase (Fig. 2B). The pattern of change in the DNMT3b mRNA levels was similar to that for DNMT3a, but the changes were not statistically significant (Fig. 2C).

Figure 2

Changes in mRNA levels of DNMT1 (A), DNMT3a (B) and DNMT3b (C) in the human endometrium during the menstrual cycle. Total RNA was isolated from endometrial tissues and subjected to real-time RT–PCR. Endometrial tissues were obtained from the proliferative phase (n = 14), early secretory phase (n = 10), mid-secretory phase (n = 11) and late secretory phase (n = 7). Relative mRNA expression normalized to TBP (internal control) was calculated. Values are mean ± SD. Different letters indicate significant differences between groups (P < 0.01 in A, P < 0.05 in B).

Figure 2

Changes in mRNA levels of DNMT1 (A), DNMT3a (B) and DNMT3b (C) in the human endometrium during the menstrual cycle. Total RNA was isolated from endometrial tissues and subjected to real-time RT–PCR. Endometrial tissues were obtained from the proliferative phase (n = 14), early secretory phase (n = 10), mid-secretory phase (n = 11) and late secretory phase (n = 7). Relative mRNA expression normalized to TBP (internal control) was calculated. Values are mean ± SD. Different letters indicate significant differences between groups (P < 0.01 in A, P < 0.05 in B).

Since mRNA levels of DNMT1, DNMT3a and DNMT3b in the endometrium were lower in the mid-secretory phase than in the proliferative phase, we examined whether DNMT mRNA expression is influenced by progesterone and estrogen. We therefore focused on ESC, which differentiate to decidualized stromal cells under the influence of progesterone and estrogen during the secretory phase. In order to induce decidualization in vitro, ESC were treated with MPA and estradiol for 17 days. As shown in Fig. 3A, mRNA expression of IGFBP-1, a specific marker of decidualization, was clearly induced by MPA and estradiol for 17 days. DNMT3a and DNMT3b mRNA levels were gradually decreased by MPA + estradiol and were significantly lower in the MPA + estradiol group than in the control group on days 8 and 17 after treatment (Fig. 3C and D). However, DNMT1 mRNA levels did not change during the treatment (Fig. 3B).

Figure 3

Effects of progesterone and estrogen on mRNA expression of DNMT1 (B), DNMT3a (C) and DNMT3b (D) in cultured ESC. Tissues were obtained from three individuals, and the cells from an individual were cultured in triplicate. Cells were treated with estradiol (E, 10−8 M) and MPA (10−6 M) for 17 days to induce decidualization in vitro. Decidualization was confirmed by mRNA expression of IGFBP-1, which is a specific marker of decidualization (A). Total RNA was isolated from cultured cells, and real-time RT–PCR for DNMTs or IGFBP-1 was performed and relative mRNA expression was calculated as described in Materials and Methods section. Values are mean ± SEM of three different cultures. Different letters indicate significant differences between groups (P< 0.05 in A and D, P < 0.01 in C).

Figure 3

Effects of progesterone and estrogen on mRNA expression of DNMT1 (B), DNMT3a (C) and DNMT3b (D) in cultured ESC. Tissues were obtained from three individuals, and the cells from an individual were cultured in triplicate. Cells were treated with estradiol (E, 10−8 M) and MPA (10−6 M) for 17 days to induce decidualization in vitro. Decidualization was confirmed by mRNA expression of IGFBP-1, which is a specific marker of decidualization (A). Total RNA was isolated from cultured cells, and real-time RT–PCR for DNMTs or IGFBP-1 was performed and relative mRNA expression was calculated as described in Materials and Methods section. Values are mean ± SEM of three different cultures. Different letters indicate significant differences between groups (P< 0.05 in A and D, P < 0.01 in C).

Discussion

The present study showed changes in the level of DNMT mRNAs in the human endometrium during the menstrual cycle. DNMT3a mRNA levels were significantly lower in the secretory phase than in the proliferative phase, being lowest in the mid-secretory phase. The pattern of change in DNMT3b mRNA levels was similar to that for DNMT3a. Furthermore, we showed that DNMT3a and DNMT3b mRNA level in ESC was down-regulated by MPA and estrogen. DNMT3a and DNMT3b are responsible for de novo CpG methylation (Hsieh, 1999). CpG methylation of the promoter region inactivates gene expression (Nan et al., 1998; Ushijima and Okochi-Takada, 2005). These findings lead us to speculate that the down-regulation of DNMT3a and DNMT3b mRNAs may be associated with expression of the genes that are induced during decidualization. In fact, a great number of genes are up-regulated and some genes are newly expressed in the human endometrium undergoing decidualization (Popovici et al., 2000; Kao et al., 2002; Riesewijk et al., 2003; Ace and Okulicz, 2004; Mirkin et al., 2005). Further studies are needed to find out which genes are regulated by DNA methylation during decidualization.

In the present study, DNMT1 mRNA levels in the endometrial tissue were significantly lower in the mid-secretory than in the proliferative phase, whereas DNMT1 mRNA levels were not affected by MPA and estradiol in ESC undergoing decidualization. Therefore, the low levels of DNMT1 in the mid-secretory phase endometrium may reflect the levels in the endometrial epithelium rather than the levels in the ESC. DNMT1 is responsible for accurately replicating genomic DNA methylation patterns to maintain genome stability during cell division (Liu et al., 1998). Endometrial epithelial cells do not proliferate during the mid-secretory phase, which seems to be compatible with the decreased DNMT1 expression during the mid-secretory phase.

There seems to be a discrepancy in this study between mRNA and protein levels for DNMT, and this may be due to the different sensitivities of RT–PCR and immunohistochemistry.

Little information is available regarding the regulation of DNMT expression. Interestingly, the present study showed that DNMT3a and DNMT3b are under the regulation of female sex steroid hormones, suggesting that DNA methylation may be influenced by female sex steroid hormones. It has been reported that DNA methylation status can be altered by a variety of factors including steroids and vitamins (Shiota, 2004). On the other hand, DNA methylation affects estrogen receptors in endometria, mammary glands and myometrium (Lapidus et al., 1996; Iwase et al., 1999; Giacinti et al., 2006; Asada et al., 2008). These findings suggest a close relationship between DNA methylation and female sex steroid hormones. However, further study is needed to clarify the molecular mechanisms of regulation of DNMTs expression.

The phase-specific and transient changes in the DNMT mRNAs during the menstrual cycle may suggest that DNA methylation status is changeable during the menstrual cycle, which may lead to changes in transcription levels of some genes. This is supported by recent reports that DNMTs are involved in both methylation and demethylation of CpG dinucleotides in human cells with cyclical changes in DNA methylation status (Kangaspeska et al., 2008; Metivier et al., 2008).

This study showed that DNMT mRNA levels change in the human endometrium during the menstrual cycle and that DNMT3a and DNMT3b mRNAs in ESC can be regulated by female sex steroid hormones. These results suggest that DNMTs have regulatory functions on gene expression in the human endometrium. Further studies are needed to show a potential role of epigenetic regulation in gene expression that is associated with decidualization.

Authors contribution

Y.Y.: conception and design, acquisition of data, analysis of data and drafting the article; H.A., L.L., I.T., R.M., K.T., T.T., A.M. and H.T.: acquisition of data and N.S.: conception and design, interpretation of data, drafting the article and final approval.

Funding

This work was supported in part by Grants-in-Aid 17791121, 18791158, 19791153 and 20591918 for Scientific Research from the Ministry of Education, Science, and Culture, Japan.

References

Ace
CI
Okulicz
WC
Microarray profiling of progesterone-regulated endometrial genes during the rhesus monkey secretory phase
Reprod Biol Endocrinol
 , 
2004
, vol. 
2
 pg. 
54
 
Asada
H
Yamagata
Y
Taketani
T
Matsuoka
A
Tamura
H
Hattori
N
Ohgane
J
Hattori
N
Shiota
K
Sugino
N
Potential link between estrogen receptor-α gene hypomethylation and uterine fibroid formation
Mol Hum Reprod
 , 
2008
, vol. 
14
 (pg. 
539
-
545
)
Esteller
M
Fraga
MF
Paz
MF
Campo
E
Colomer
D
Novo
FJ
Calasanz
MJ
Galm
O
Guo
M
Benitez
J
, et al.  . 
Cancer epigenetics and methylation
Science
 , 
2002
, vol. 
297
 (pg. 
1807
-
1808
)
Giacinti
L
Claudio
PP
Lopez
M
Giordano
A
Epigenetic information and estrogen receptor alpha expression in breast cancer
Oncologist
 , 
2006
, vol. 
11
 (pg. 
1
-
8
)
Girault
I
Tozlu
S
Lidereau
R
Bieche
I
Expression analysis of DNA methyltransferases 1, 3A, and 3B in sporadic breast carcinomas
Clin Cancer Res
 , 
2003
, vol. 
9
 (pg. 
4415
-
4422
)
Giudice
LC
Dsupin
BA
Irwin
JC
Steroid and peptide regulation of insulin-like growth factor-binding proteins secreted by human endometrial stromal cells is dependent on stromal differentiation
J Clin Endocrinol Metab
 , 
1992
, vol. 
75
 (pg. 
1235
-
1241
)
Hsieh
CL
In vivo activity of murine de novo methyltransferases, DNMT3a and DNMT3b
Mol Cell Biol
 , 
1999
, vol. 
19
 (pg. 
8211
-
8218
)
Imamura
T
Ohgane
J
Ito
S
Ogawa
T
Hattori
N
Tanaka
S
Shiota
K
CpG island of rat sphingosine kinase-1 gene: tissue-dependent DNA methylation status and multiple alternative first exons
Genomics
 , 
2001
, vol. 
76
 (pg. 
117
-
125
)
Iwase
H
Omoto
Y
Iwata
H
Toyama
T
Hara
Y
Ando
Y
Ito
Y
Fujii
Y
Kobayashi
S
DNA methylation analysis at distal and proximal promoter regions of the oestrogen receptor gene in breast cancer
Br J Cancer
 , 
1999
, vol. 
80
 (pg. 
1982
-
1986
)
Izawa
M
Harada
T
Taniguchi
F
Ohama
Y
Takenaka
Y
Terakawa
N
An epigenetic disorder may cause aberrant expression of aromatase gene in endometriotic stromal cells
Fertil Steril
 , 
2008
, vol. 
89
 (pg. 
1390
-
1396
)
Jones
PA
Baylin
SB
The fundamental role of epigenetic events in cancer
Nature Rev
 , 
2002
, vol. 
3
 (pg. 
415
-
428
)
Kangaspeska
S
Stride
B
Metivier
R
Polycarpou-Schwarz
M
Ibberson
D
Carmouche
RP
Benes
V
Gannon
F
Reid
G
Transient cyclical methylation of promoter DNA
Nature
 , 
2008
, vol. 
452
 (pg. 
112
-
115
)
Kao
LC
Tulac
S
Lobo
S
Imani
B
Yang
JP
Germeyer
A
Osteen
K
Taylor
RN
Lessey
BA
Giudice
LC
Global gene profiling in human endometrium during the window of implantation
Endocrinology
 , 
2002
, vol. 
143
 (pg. 
2119
-
2138
)
Lapidus
RG
Ferguson
AT
Ottaviano
YL
Parl
FF
Smith
HS
Weitzman
SA
Baylin
SB
Issa
J-P
Davidson
NE
Methylation of estrogen and progesterone receptor gene 5′ CpG islands correlates with lack of estrogen and progesterone receptor gene expression in breast tumors
Clin Cancer Res
 , 
1996
, vol. 
2
 (pg. 
805
-
810
)
Li
E
Chromatin modification and epigenetic reprogramming in mammalian development
Nat Rev Genet
 , 
2002
, vol. 
3
 (pg. 
662
-
673
)
Li
E
Beard
C
Jaenisch
R
Role for DNA methylation in genomic imprinting
Nature
 , 
1993
, vol. 
366
 (pg. 
362
-
365
)
Liu
Y
Okakeley
EJ
Sun
L
Jost
JP
Multiple domains are involved in the targeting of the mouse DNA methyltransferase to the DNA replication foci
Nucleic Acids Res
 , 
1998
, vol. 
26
 (pg. 
1038
-
1045
)
Metivier
R
Gallais
R
Tiffoche
C
Le Peron
C
Jurkowska
RZ
Carmouche
RP
Ibberson
D
Barath
P
Demay
F
Reid
G
, et al.  . 
Cyclical DNA methylation of a transcriptionally active promoter
Nature
 , 
2008
, vol. 
452
 (pg. 
45
-
50
)
Mirkin
S
Arslan
M
Churikov
D
Corica
A
Diaz
JI
Williams
S
Bocca
S
Oehninger
S
In search of candidate genes critically expressed in the human endometrium during the window of implantation
Hum Reprod
 , 
2005
, vol. 
20
 (pg. 
2104
-
2117
)
Nan
X
Ng
H-H
Johnson
CA
Laherty
CD
Turner
BM
Eisenman
RN
Bird
A
Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex
Nature
 , 
1998
, vol. 
393
 (pg. 
386
-
389
)
Noyes
RW
Hertig
AT
Rock
J
Dating the endometrial biopsy
Fertil Steril
 , 
1950
, vol. 
1
 (pg. 
3
-
25
)
Ohgane
J
Aikawa
J
Ogura
A
Hattori
N
Ogawa
T
Shiota
K
Analysis of CpG islands of trophoblast giant cells by restriction landmark genomic scanning
Dev Genet
 , 
1998
, vol. 
22
 (pg. 
132
-
140
)
Okada
H
Nakajima
T
Yoshimura
T
Yasuda
K
Kanzaki
H
Microarray analysis of genes controlled by progesterone in human endometrial stromal cells
Gynecol Endocrinol
 , 
2003
, vol. 
17
 (pg. 
271
-
280
)
Ponnampalam
AP
Weston
GC
Trajstman
AC
Susil
B
Rogers
PAW
Molecular classification of human endometrial cycle stages by transcriptional profiling
Mol Hum Reprod
 , 
2004
, vol. 
10
 (pg. 
879
-
893
)
Popovici
RM
Kao
LC
Giudice
LC
Discovery of new inducible genes in in vitro decidualized human endometrial stromal cells using microarray technology
Endocrinology
 , 
2000
, vol. 
141
 (pg. 
3510
-
3513
)
Razin
A
Kafri
T
DNA methylation from embryo to adult
Prog Nucleic Acid Res Mol Biol
 , 
1994
, vol. 
48
 (pg. 
53
-
81
)
Riesewijk
A
Martin
J
Os
RV
Horcajadas
JA
Polman
J
Pellicer
A
Mosselman
S
Simon
C
Gene expression profiling of human endometrial receptivity on days LH+2 versus LH+7 by microarray technology
Mol Hum Reprod
 , 
2003
, vol. 
9
 (pg. 
253
-
264
)
Sakai
N
Maruyama
T
Sakurai
R
Masuda
H
Yamamoto
Y
Shimizu
A
Kishi
I
Asada
H
Yamagoe
S
Yoshimura
Y
Involvement of histone acetylation in ovarian steroid-induced decidualization of human endometrial stromal cells
J Biol Chem
 , 
2003
, vol. 
278
 (pg. 
16675
-
16682
)
Shiota
K
DNA methylation profiles of CpG islands for cellular differentiation and development in mammals
Cytogenet Genome Res
 , 
2004
, vol. 
105
 (pg. 
325
-
334
)
Shiota
K
Yanagimachi
R
Epigenetics by DNA methylation for development of normal and cloned animals
Differentiation
 , 
2002
, vol. 
69
 (pg. 
162
-
166
)
Smith
SS
Biological implications of the mechanism of action of human DNA (cytosine-5)methyltransferase
Prog Nucleic Acid Res Mol Biol
 , 
1994
, vol. 
49
 (pg. 
65
-
111
)
Sugino
N
Shimamura
K
Takiguchi
S
Tamura
H
Ono
M
Nakata
M
Nakamura
Y
Ogino
K
Uda
T
Kato
H
Changes in activity of superoxide dismutase in the human endometrium throughout the menstrual cycle and in early pregnancy
Hum Reprod
 , 
1996
, vol. 
11
 (pg. 
1073
-
1078
)
Sugino
N
Kashida
S
Takiguchi
S
Nakamura
Y
Kato
H
Induction of superoxide dismutase by decidualization in human endometrial stromal cells
Mol Hum Reprod
 , 
2000
, vol. 
6
 (pg. 
178
-
184
)
Sugino
N
Karube-Harada
A
Kashida
S
Takiguchi
S
Kato
H
Differential regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase by progesterone withdrawal in human endometrial stromal cells
Mol Hum Reprod
 , 
2002
, vol. 
a 8
 (pg. 
68
-
74
)
Sugino
N
Kashida
S
Karube-Harada
A
Takiguchi
S
Kato
H
Expression of vascular endothelial growth factor and its receptors in the human endometrium throughout the menstrual cycle and in early pregnancy
Reproduction
 , 
2002
, vol. 
b 123
 (pg. 
379
-
387
)
Sugino
N
Karube-Harada
A
Sakata
A
Takiguchi
S
Kato
H
Nuclear factor-κB is required for tumor necrosis factor-alpha induced manganese superoxide dismutase expression in human endometrial stromal cells
J Clin Endocrinol Metab
 , 
2002
, vol. 
c 87
 (pg. 
3845
-
3850
)
Sugino
N
Karube-Harada
A
Sakata
A
Takiguchi
S
Kato
H
Different mechanisms for the induction of copper-zinc superoxide dismutase and manganese superoxide dismutase by progesterone in human endometrial stromal cells
Hum Reprod
 , 
2002
, vol. 
d 17
 (pg. 
1709
-
1714
)
Sugino
N
Karube-Harada
A
Taketani
T
Sakata
A
Nakamura
Y
Withdrawal of ovarian steroids stimulates prostaglandin F2α production through nuclear factor-κB activation via oxygen radicals in human endometrial stromal cells: potential relevance to menstruation
J Reprod Dev
 , 
2004
, vol. 
50
 (pg. 
215
-
225
)
Ting
AH
Jair
KW
Suzuki
H
Yen
RW
Baylin
SB
Schuebel
KE
Mammalian DNA methyltransferase 1: inspiration for new directions
Cell Cycle
 , 
2004
, vol. 
3
 (pg. 
1024
-
1026
)
Uchida
H
Maruyama
T
Nagashima
T
Asada
H
Yoshimura
Y
Histone deacetylase inhibitors induce differentiation of human endometrial adenocarcinoma cells through up-regulation of glycodelin
Endocrinology
 , 
2005
, vol. 
146
 (pg. 
5365
-
5373
)
Ushijima
T
Okochi-Takada
E
Aberrant methylations in cancer cells: where do they come from?
Cancer Sci
 , 
2005
, vol. 
96
 (pg. 
206
-
211
)
Utsunomiya
H
Cheng
YH
Lin
Z
Reierstad
S
Yin
P
Attar
E
Xue
Q
Imir
G
Thung
S
Trukhacheva
E
, et al.  . 
Upstream stimulatory factor-2 regulates steroidogenic factor-1 expression in endometriosis
Mol Endocrinol
 , 
2008
, vol. 
22
 (pg. 
904
-
914
)
Wu
Y
Halverson
G
Basir
Z
Strawn
E
Yan
P
Guo
SW
Aberrant methylation at HOXA10 may be responsible for its aberrant expression in the endometrium of patients with endometriosis
Am J Obstet Gynecol
 , 
2005
, vol. 
193
 (pg. 
371
-
380
)
Wu
Y
Strawn
E
Basir
Z
Halverson
G
Guo
SW
Aberrant expression of deoxyribonucleic acid methyltransferases DNMT1, DNMT3A, and DNMT3B in women with endometriosis
Fertil Steril
 , 
2007
, vol. 
87
 (pg. 
24
-
32
)
Xue
Q
Lin
Z
Cheng
YH
Huang
CC
Marsh
E
Yin
P
Milad
MP
Confino
E
Reierstad
S
Innes
J
, et al.  . 
Promoter methylation regulates estrogen receptor 2 in human endometrium and endometriosis
Biol Reprod
 , 
2007
, vol. 
a 77
 (pg. 
681
-
687
)
Xue
Q
Lin
Z
Yin
P
Milad
MP
Cheng
YH
Confino
E
Reierstad
S
Bulun
SE
Transcriptional activation of steroidogenic factor-1 by hypomethylation of the 5′ CpG island in endometriosis
J Clin Endocrinol Metab
 , 
2007
, vol. 
b 92
 (pg. 
3261
-
3267
)