Increased Expression and Altered Methylation of

HERVWE1 in the Human Placentas of Smaller Fetuses

from Monozygotic, Dichorionic, Discordant Twins
Yu Gao1,2., Zhiming He1,2., Zilian Wang 1 , Yanmin Luo 1 , Hongyu Sun3, Yi Zhou1, Linhuan Huang1 ,
Manchao Li 1, Qun Fang 1 *, Shiwen Jiang2,4*
1 Fetal Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guang Dong Province, People’s
Republic of China, 2 Hoskins Center, Department of Biological Science, Mercer University School of Medicine, Savannah, Georgia, United States of America, 3 Department
of Forensic Medicine, Sun Yat-Sen University, Guangzhou, Guang Dong Province, People’s Republic of China, 4 Department of Obstetrics and Gynecology, Mayo College of
Medicine, Rochester, Minnesota, United States of America

Abstract
Background: The human endogenous retroviral family W, Env(C7), member 1 gene (HERVWE1) is thought to participate in
trophoblast cell fusion, and its expression is diminished in the placentas of singleton intrauterine growth-retarded
pregnancies. However, there is limited information about the role of HERVWE1 in discordant fetal growth in twins. This study
was to compare HERVWE1 gene expression between the placentas of discordant monozygotic twins and to identify its
regulation by methylation.

Methodology/Principal Findings: Fetuses from twenty-one pairs of monozygotic, dichorionic, discordant twins were
marked as ‘‘smaller’’ or ‘‘larger’’ according to birth weight. Placental HERVWE1 mRNA and protein expression profiles were
analyzed using quantitative RT-PCR and immunohistochemistry (IHC) staining. Methylation profiles of the HERVWE1
promoter region were analyzed using a pyrosequencing assay. DNA methyltransferase (DNMT) transcript levels were
analyzed by RT-PCR. 5-methyl cytosine (5-MC) was stained using an immunohistochemical assay. There was a significant
negative correlation between HERVWE1 mRNA levels and birth weight in twins (P,0.01). Whereas the mean methylation
level of the HERVWE1 promoter region was diminished in the smaller group in discordant twins(P,0.01), increased mRNA
and protein levels of HERVWE1 were found in smaller fetuses compared with larger fetuses in discordant twins(P,0.01).
There was no significant difference in 5-MC staining intensity between discordant twins (P.0.05). The DNMT3b3 mRNA
levels in the smaller group were significantly downregulated compared with the larger group in discordant twins(P,0.05),
whereas the DNMT3b7 mRNA levels in the smaller group were significantly upregulated compared with the larger group in
discordant twins(P,0.05).

Conclusions/Significance: In discordant, monozygotic, dichorionic twins, HERVWE1 expression was higher in smaller fetuses
and lower in larger fetuses. Methylation of the HERVWE1 gene promoter region may participate in the regulation of
HERVWE1 gene expression in discordant twin pregnancies.

Citation: Gao Y, He Z, Wang Z, Luo Y, Sun H, et al. (2012) Increased Expression and Altered Methylation of HERVWE1 in the Human Placentas of Smaller Fetuses
from Monozygotic, Dichorionic, Discordant Twins. PLoS ONE 7(3): e33503. doi:10.1371/journal.pone.0033503
Editor: Pierre-Antoine Defossez, Université Paris-Diderot, France
Received September 20, 2011; Accepted February 10, 2012; Published March 21, 2012
Copyright: ß 2012 Gao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: No current external funding sources received for this study.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected] (QF); [email protected] (SJ)
. These authors contributed equally to this work.

Introduction functions in the placenta, including fusion, proliferation, anti-

apoptosis, and immune suppression [3,4]. The fusion function of
The placenta plays an important role in fetal growth and syncytin-1 promotes the merging of cytotrophoblast cells to form
development. Its key structure, a syncytium, maintains maternal- the syncytiotrophoblast cell, which secretes several endocrine
fetal nutrient transport and releases hormones. The structure of hormones that promote fetal growth, such as human chorionic
the syncytium in the placenta undergoes significant changes during gonadotrophin (HCG) and human chorionic somatomammotro-
intrauterine growth retardation (IUGR) [1]. Over the past few pin (HCS). HERVWE1 expression is regulated by transcription
years, researchers have found that human endogenous retroviral factors, hormones, cytokines, environmental conditions, and DNA
family W, Env (C7), member 1 (HERVWE1), which is highly methylation. Chen and colleagues have proven that DNA
expressed in placental tissue, is the critical gene that regulates the methylation is one of the most important mechanisms for
action and preservation of the syncytium [1,2]. The protein regulating HERVWE1 [5]. Matouskova and Gimenez have shown
product of HERVWE1 is syncytin-1, which mediates special that promoter region hypermethylation diminished HERVWE1

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 HERVWE1 Expression, Methylated Regulation in Twins

transcript levels, whereas hypomethylation enhanced its transcript capillary electrophoresis assay. Among those pairs, there were 21
levels in placental and several non-placental tissues [6,7]. Aside pairs of discordant twins for whom the birth weight difference was
from the gene-specific promoter region methylation profile, global at least 20%. All 42 infants were enrolled into the larger or smaller
genomic methylation and the key enzymes regulating DNA group by pair according to birth weight. All of these cases were
methylation (DNA methyltransferases, DNMTs) may affect gene managed expectantly without any interventions. In addition, we
expression. In the last five years, HERVWE1 expression has been collected data from 10 cases of singleton pregnancies delivered at
found to be suppressed in IUGR placentas [8,9]. However, 30–36 weeks for the singleton control group,and 24 pairs of
previous research has focused on singleton pregnancies. The concordant monozygotic dichorionic twins delivered at 30–36
evidence in multiple pregnancies remains limited. weeks for the twins control group, for whom the birth weight
Monozygotic twins have identical inherited backgrounds and difference was less than 20%. All the cases included in this study
similar intrauterine circumstances. However, phenotypic discrep- were delivered by cesarean section. All of the patients’ parents or
ancies generally do exist between monozygotic twins [10,11]. For guardians were informed about the following research data
example, birth weight discordance, which is defined as an inter- collection. Consent was signed prior to enrollment. This study
twin birth weight difference of greater than 20%, is one abnormal was approved by the Sun Yat-sen University Institutional Review
twin fetal growth pattern [12,13]. An unbalanced placental blood Board.
supply caused by vessel anastomosis is one of the most important
pathogeneses leading to weight discordance in monozygotic, Sample collection
monochorionic twins [14,15]. However, this cannot explain the Within 15 minutes after delivery, placental tissue from around
discordance in monozygotic, dichorionic twins as these twins share the individual insertion region of the umbilical cord was collected.
the same DNA sequence and similar circumstances, and there are The placental tissue was excised from inside the placental lobules,
seldom communicating branch vessels in the placenta. Unequal avoiding both the maternal surface and the amniotic membrane.
placental sharing has been identified in some discordant weight Three 262 cm tissue samples were excised and washed 3 times in
cases due to a lack of anastomoses [16]. However, the details of sterilized, ice-cold saline to eliminate any blood. One piece of
this molecular mechanism remain unclear. tissue was placed in TRIzol solution for RNA isolation. The
Some researchers have studied the phenotypic differences second piece of tissue was placed in a cryotube, which was then
between adult monozygotic twins and found that epigenetic deep frozen in liquid nitrogen overnight and transferred to a
modification is one of the most important causes [17–20]. 280uC freezer for storage prior to DNA isolation. The third piece
However, most of these studies focused on adult diseases, such of tissue was placed in a 10% formalin solution overnight and
as psychiatric disorders [11,21,22], multiple sclerosis [20,23], and embedded in paraffin for future immunohistochemical staining.
systemic lupus erythematosus [24], and the study specimens were
peripheral blood or epithelial cells. Few studies have examined
RNA/DNA extraction and conversion
intrauterine epigenetic modification in the placenta of discordant,
RNA isolation was performed using TRIzol (Invitrogen, Cat.
monozygotic twins. Moreover, Fraga and coworkers pointed out
No. 15596-018, Carlsbad, CA, USA) and the phenol-chloroform
that it takes a long time to accumulate significant changes in
method. The RNA was treated with Ambion DNA-free DNase
epigenetic modification [25]. It is still unclear whether epigenetic
treatment and removal reagents (Applied Biosystems, Part
regulation affects fetal growth discordance during early life.
No. AM1906, Austin, TX, USA). The RNA was reverse
In this study, we investigated whether differential expression of
transcribed to cDNA using a High Capacity RNA-to-cDNA kit
HERVWE1 and/or methylation of its promoter region were
(Applied Biosystems, Part No. 4387406, Austin, TX, USA). The
related to differences in the birth weight of twins, using gene
cDNA was stored in a 220uC freezer until use in the real-time
expression and methylation analyses to examine placentas from
PCR assay.
monozygotic, dichorionic, discordant twins. In addition, we
DNA was extracted using the phenol-chloroform method.
hypothesized that the key enzymes that regulate DNA methylation
Bisulfite conversion was achieved with an EpiTect Bisulfite Kit
(DNMTs) contribute to the alterative methylation profile of the
(Qiagen, Cat No 59104, Valencia, CA, USA). Bisulfite-treated
HERVWE1 promoter region in monozygotic, dichorionic, discor-
DNA was stored in a 220uC freezer until use in the
dant twins. As far as we are aware, this study is the first to use
pyrosequencing assay.
placenta samples from monozygotic, dichorionic twins to study
HERVWE1. Our findings may increase our understanding of the
role of the placenta in maintaining normal fetal growth in twin Zygosity identification using a capillary electrophoresis
pregnancies. assay
Genomic DNA was amplified with PowerPlex 16 (Promega, Cat
Material and Methods No. DC 6531, Madison, WI, USA) in order to detect 15
autosomal short tandem repeat (STR) loci in addition to the
Case enrollment gender determination marker amelogenin. Multiplex PCR was
All of the data for the twin pregnancies were collected at the performed using 1 ml of template DNA in a 10 ml volume that
First Affiliated Hospital of Sun Yat-Sen University, China, from included 1 ml of 106 GeneAmp PCR Gold Buffer, 1 ml of 106
July 2003 to December 2009. All intrauterine fetal deaths, IUGR, Primer Pair Mix, and 0.3 ml of AmpliTaq Gold DNA polymerase
twin-twin transfusion syndromes (TTTS), and infants delivered (1.5 U) (Applied Biosystems, Cat No. 4338856, Carlsbad, CA,
before 26 gestational weeks were excluded from the study. USA). The PCR cycling conditions were 95uC for 11 minutes;
Chorionicity was determined by a pathology exam after delivery. 96uC for 2 minutes; 10 cycles of 94uC for 1 minute, 60uC for
The total number of dichorionic twin pregnancies was 336. 1 minute, and 70uC for 1.5 minutes; 22 cycles of 90uC for
Among these cases, 134 pairs of different-sex twins were excluded 1 minute, 60uC for 1 minute, and 70uC for 1.5 minutes; and a
due to dizygosity. Zygosity identification was applied in the cases final extension at 60uC for 30 minutes. The PCR products were
of 202 pairs of same-sex twins. A total of 56 pairs were identified as separated and detected by capillary electrophoresis using an ABI
monozygotic twins based on zygosity identification using a 3100 Genetic Analyzer (Applied Biosystem, Cat No. 4359571,

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 HERVWE1 Expression, Methylated Regulation in Twins

Carlsbad, CA, USA) according to the manufacturer’s instructions. reaction contained 6 ml 26 SYBR green master mixture, 1 ml
Allele calls were made with GeneMapper ID v3.2 software forward primer, 1 ml reverse primer, 1 ml cDNA template, and
(Applied Biosystem, Cat No. 4338856, Carlsbad, CA, USA). If all 3 ml H2O. Every reaction was repeated in quadruplicate. The
genotypes of the 16 loci were identical, then the pair was identified thermal cycling conditions consisted of 50uC for 2 minutes; 95uC
as monozygotic. If the genotypes of any loci were different, then for 6 minutes; and 40 cycles of 95uC for 30 seconds, 45uC for
the pair was identified as dizygotic. Appendix Figure S1 shows the 30 seconds, and 60uC for 1 minute. The quantitative gene
16 STR loci in one pair of monozygotic twins. expression results were analyzed with sequence detection system
software (SDS v.2.3). The details of the real-time primers for
Real-time PCR HERVWE1, HCS, DNMTs, GAPDH, b-actin, and PCNA are shown
To determine the transcriptional profile of HERVWE1, in Appendix Table S1.
quantitative real-time PCR was used to compare HERVWE1
mRNA levels between growth discordant twins, with three internal Immunohistochemical staining (IHC)
reference genes, glyceraldehyde-3-phosphate dehydrogenase Blocks from ten pairs of discordant twins, ten pairs of
(GAPDH),beta actin (b-actin), and proliferating cell nuclear antigen concordant twins, and ten singleton cases were available to apply
(PCNA).. The HCS gene located downstream of HERVWE1 was the HERVWE1 IHC staining. The same ten pairs of discordant
identified as a factor reflecting placental function. The HCS twins and ten singleton cases applied the 5-MC staining. The slices
mRNA level was measured as well. The concordant twins groups were stained under the same condition in HERVWE1 IHC
and singleton group served as normal controls. We also explored staining and 5-MC staining respectively.
the transcription of three DNMTs (DNMT1, DNMT3a, and
DNMT3b) and seven DNMT3b isoforms (DNMT3b1-7) in discor- HERVWE1 protein expression
dant twins and singleton control group using real-time PCR. Placental tissue was embedded in paraffin. Each slice was
Briefly, cDNA was quantitatively analyzed by real-time PCR deparaffinized using xylene and gradient ethanol. Antigen retrieval
using a 7900HT fast real-time PCR system (Applied Biosystems, was performed in boiling 10 mM citrate buffer for 10 minutes.
Part No. 4329001, Carlsbad, CA, USA). PCR was performed in The slices were then treated with 2 N hydrochloric acid (HCl) at
384-well plates using SYBR Green PCR Master Mix (Applied 37uC for 30 minutes. Endo-peroxidase blocking was accomplished
Biosystems, Part No. 4309155, Carlsbad, CA, USA). Each with 3% H2O2 in methanol for 20 minutes. Blocking of non-

Table 1. Clinical characteristics among the groups.

Larger twin in Smaller twin in Larger twin in Smaller twin in

discordant group discordant group concordant group concordant group Singleton group
(n = 21) (n = 21) (n = 24) (n = 24) (n = 10)

Maternal age (years) 27.9564.22 28.3863.98 27.6062.07

Race
Asian (n, %) 21 (100%) 24 (100%) 10 (100%)
Non-Asian (n, %) 0 (0%) 0 (0%) 0 (0%)
Maternal BMI (kg/m2) 24.663.8 24.263.6 22.163.2
Maternal parity 1.4560.6 1.2960.6 1.5660.7
Mode of delivery
Vaginal delivery (n, %) 0 (0%) 0 (0%) 0 (0%)
Cesarean section (n, %) 21 (100%) 24 (100%) 10 (100%)
Delivery GA (weeks) 32.8762.57 33.6962.08 33.4162.43
Sex distribution
Male (n, %) 16 (76.2%) 14 (58.3%) 6 (60%)
Female (n, %) 5 (23.8%) 10 (41.7%) 4 (40%)
Maternal complications
Preeclampsia (n, %) 0 (0%) 0 (0%) 0 (0%)
TTTS (n, %) 0 (0%) 0 (0%) _
IUGR (n, %) 0 (0%) 0 (0%) 0 (0%)
GDM (n, %) 0 (0%) 0 (0%) 0 (0%)
Birth weight (kg) 1.9060.43* 1.3760.36{{ 2.0660.37 1.886.0.36 2.0760.44
Birth weight difference (%) 28.565.3j 8.765.6 _

Data are shown as the means 6 standard deviations or n (%).

BMI, body mass index; GA, gestational age; TTTS, twin-to-twin transfusion syndrome; IUGR, intrauterine growth restriction.
*P,0.01 vs. smaller twin in discordant group.
{P,0.01 vs. singleton group.
{P,0.01 vs. larger or smaller twins in concordant group.
j
P,0.05 vs. concordant twin group.
doi:10.1371/journal.pone.0033503.t001

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 HERVWE1 Expression, Methylated Regulation in Twins

specific binding was performed with 1% gelatin. The samples were Appendix Figure S2 is a schematic diagram showing the CG sites
incubated at 4uC overnight with a 1:100 dilution of affinity-purified in the HERVWE1 promoter region. To analyze the methylation
rabbit polyclonal anti-human endogenous retrovirus IgG antibody levels of the 5 CG sites, we used a pyrosequencing assay. The
(GeneTex Inc. Cat No. GTX70327, Irvine, CA, USA). A goat anti- bisulfite-converted target DNA sequence was amplified using biotin-
rabbit secondary antibody conjugated to biotin (Vector Laboratories, labeled primers, as shown in Appendix Table S1. Each PCR
Cat No. BA-1000, Burlingame, CA, USA) at a dilution of 1:200 was reaction contained 3 ml 106 PCR buffer, 1.8 ml 25 mM/L MgCl2,
applied for 30 minutes. The sections were then incubated with a 0.6 ml 10 mM dNTP (Fermentas, Cat No. R0191, Glen Burnie,
Vectastain Elite ABC-peroxidase kit (Vector laboratories, Cat MD, USA), 0.15 ml Hotstar Taq DNA polymerase (Qiagen, Cat
No. BA-1000, Burlingame, CA, USA) for 60 minutes. Staining was No. 203203, Valencia, CA, USA), 0.6 ml forward primer, 0.6 ml
detected with a substrate solution of diaminobenzidine tetrahy- reverse primer, 1 ml DNA template, and 22.25 ml H2O. The
drochloride (DAB) (Sigma Aldrich, Cat. No D5905-50, St. Louis, thermal cycling conditions consisted of 95uC for 15 minutes; 45
MO, USA). Counterstaining was performed with hematoxylin. cycles of 95uC for 30 seconds, 58uC for 30 seconds, and 72uC for
30 seconds; 72uC for 10 minutes; and 4uC until completion. The
Global methylation staining for 5-methyl cytosine (5-MC) PCR products were sent to EpigenDx, Inc. (Worcester, MA, USA)
Tissue slices were incubated overnight in a 1:200 dilution of for pyrosequencing. All new data had been deposited in GenBank.
sheep anti-5-Methyl Cytosine primary antibody (Capralogics, Inc., Because pyrosequencing can read only 50–100 bp for each accurate
Cat No. P00704, Hardwick, MA, USA) followed by a 1:200 pyrosequencing primer, we used two pyrosequencing primers to
analyze the five CpG sites. The pyrograms are shown in Appendix
dilution of biotinylated goat anti-sheep IgG (Vector Laboratories,
Figure S3. The methylation level of each CG site was calculated as
Cat No. BA-6000, Burlingame, CA, USA) as the secondary
the C/(C+T) peak height ratio. Unmethylated and in vitro
antibody. The other steps were performed as described for the
methylated DNA were mixed at specified ratios to serve as controls.
HERVWE1 staining.
Statistical analysis
Picture acquisition and processing
All of the data were analyzed using IBM SPSS v. 19.0 statistical
Images were captured using a Carl Zeiss microscope imaging
software (SPSS Inc., Chicago, IL, USA). Clinical information was
system (Carl Zeiss, Cat. No. AxioVert 200 M, Thornwood, NY,
presented as the frequencies or means 6 standard deviations. The
US). Based on the proportion of positively stained cells and their
comparisons between larger and smaller fetuses in twin pairs were
degree of intensity, three random high-power fields (10640) from
performed using an independent sample t-test. The differences
each section were analyzed using AxioVs40LE digital image
among the control groups, the smaller or larger discordant twin
processing software (Carl Zeiss, AxioVision version 4.5.0.0, groups were analyzed using one-way ANOVA. The correlations
Thornwood, NY, US). The scoring system used to assess the between methylation levels and gene expression were evaluated using
trophoblast profile was as follows: negative (score = 0) = no positive Spearman’s correlation analysis. The semi-quantitative IHC data
staining, weakly positive (score = 1) = weakly positive staining seen were analyzed using ANOVA with the least significant difference
within the structure, positive (score = 2) = positive staining seen procedure (LSD) and post hoc multiple comparisons analysis. To
within the structure, and strongly positive (score = 3) = strong remove the effect of gestational age, analysis of covariance was
staining signal within the trophoblast cell. The proportion of cells applied to evaluate the difference of gene expression in each group. A
in each staining intensity category was multiplied by the P value,0.05 was considered significant for all tests.
trophoblast profile score to calculate the overall score.
Results
Pyrosequencing assay
The pyrosequencing assay was applied in all of the discordant Clinical characteristics
twins and singleton control cases. There were 5 CG sites from There were 21 pairs of monozygotic, dichorionic, discordant
2336 bp to 2192 bp in the HERVWE1 59LTR+U3 region. twins, 24 pairs of monozygotic, dichorionic, concordant twins, and

Table 2. HERVWE1 and HCS mRNA levels among the groups (GAPDH, b-actin, and PCNA as internal reference genes respectively).

Larger twin in Smaller twin in Larger twin in Smaller twin in

discordant group discordant group concordant group concordant group Singleton group
(n = 21) (n = 21) (n = 24) (n = 24) (n = 10)

HERVWE1
GAPDH as control 1.0360.18 1.9160.31{ 0.9660.22 1.0460.29 1.0060.09
b-actin as control 0.9860.16 1.6260.29{ 1.0960.22 1.1360.26 1.0060.17
PCNA as control 0.9960.26 1.1160.27 1.0960.22 1.1260.27 1.0060.13
HCS
GAPDH as control 1.1260.33 2.1760.36{ 1.1360.32 1.1560.30 1.0060.25
b-actin as control 1.1860.29 1.7360.35{ 1.1260.31 1.1760.28 1.0060.24
PCNA as control 0.9760.34 1.0760.28 1.0860.31 1.0960.25 1.0060.24

The values shown indicate the relative quantitation of HERVWE1 and HCS mRNAs, which were standardized to GAPDH,b-actin,and PCNA respectively, and normalized to
the singleton control group.
The data are shown as the means 6 standard deviations.
{P,0.01 vs. other four groups.
doi:10.1371/journal.pone.0033503.t002

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 HERVWE1 Expression, Methylated Regulation in Twins

10 singleton pregnancies enrolled in our study. The clinical distribution among the discordant twins, concordant twins, and
characteristics are summarized in Table 1. There were no singleton groups. The birth weight of smaller fetuses in discordant
differences in maternal age, race distribution, maternal body mass group was the lightest among the five groups (P,0.01). The birth
index (BMI), maternal parity, mode of delivery, delivery weight difference in discordant twins was significantly larger than
gestational age, incidence of maternal complication, or gender that in concordant twins (P,0.05).

Figure 1. The correlation between HERVWE1 transcript levels and birth weight. The scatterplots showed the correlation between the
HERVWE1 transcript profile and the fetal birth weight in all five groups. (A) Using GAPDH as internal reference gene, the HERVWE1 transcript level was
significant negatively correlated with the fetal birth weight (spearman correlation coefficient 20.287, P,0.01). (B) Using b-actin as internal reference
gene, the HERVWE1 transcript level was significant negatively correlated with the fetal birth weight (spearman correlation coefficient 20.271,
P,0.01).
doi:10.1371/journal.pone.0033503.g001

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 HERVWE1 Expression, Methylated Regulation in Twins

Table 2, the HERVWE1 mRNA level of the smaller twin in

discordant group was greater than that of the larger twin in
discordant group (P,0.01, GAPDH and b-actin as control). The
downstream gene HCS, as an indicator of placental function, also
showed the same trend in discordant twins (P,0.05, GAPDH and
b-actin as control). The HERVWE1 and HCS transcript levels in
the larger discordant twin group were similar to that in singleton
group and two concordant twin groups (P.0.05). We observed an
association between HERVWE1 and HCS mRNA levels in the pool
of all groups, with a Spearman’s correlation coefficient (Rs) of
0.647(GAPDH as control), 0.582 (b-actin as control) and 0.624
(PCNA as control)(P,0.01). We also observed a significant negative
correlation between HERVWE1 transcript levels and birth weight
in all groups, with a correlation coefficient of 20.287 (GAPDH as
control) and 20.271 (b-actin as control) (P,0.01) (Figure 1). We
didn’t find the correlation between HERVWE1 transcript levels
and delivery gestational age in each group (P.0.05). When
gestational age was taken into account as covariate in covariance
analysis, the same increased expression was detected in smaller
fetuses in discordant twins.

HERVWE1 protein expression

HERVWE1 IHC staining images are shown in Figure 2. There
was no significant difference in pathological structure examination
among groups. All of the villous trees were misaligned and
crowded, and the stem villi were scarred. The stem villi were short
and thick. The majority of the villi were terminal villi, and they
Figure 2. HERVWE1 staining of discordant twin, concordant were tiny and irregular. The terminal villus was covered with a
twin, and singleton placentas. A–F show placental tissues with trophoblastic surface, which was composed of two layers. The
HERVWE1 immunohistochemical staining. The paraffin-embedded
placental tissues were sliced into 4-mm sections. The slices were villous syncytiotrophoblast constituted the outer continuous thin
incubated in a rabbit polyclonal anti-human endogenous retrovirus IgG layer. The villous cytotrophoblast constituted the inner discontin-
primary antibody (diluted 1:100). A goat anti-rabbit secondary antibody uous layer. Inside the villi was the stroma, including the fetal
(diluted in 1:200) was applied sequentially. The slices were developed vessels, macrophages, and connective tissue fibers. The positive
with an immunoperoxidase system. The HERVWE1 antigen was mainly staining of HERVWE1 was mostly concentrated in the cytoplasm
present in the trophoblast cytoplasm. The positive cells were stained
brown, and the negative cells were stained blue after counterstaining
of the trophoblast cells. There was no significant difference in
with hematoxylin. (A) The larger infant in discordant group (magnifi- syncytiotrophoblast or cytotrophoblast cell number among all
cation 10640). (B) The smaller infant in discordant group (magnification groups.
10640). (C) The larger infant in concordant group (magnification We compared the intensity of trophoblast cells among the
10640). (D) The smaller infant in concordant group (magnification discordant twin group, concordant twin group, and singleton
10640). (E) A singleton infant (magnification 10640). (F) The negative group (Table 3). The smaller twin in discordant group had the
control (magnification 10640). This singleton tissue slice was stained
with 1% gelatin instead of the HERVWE1 antibody. Syncytiotrophoblast, strongest HERVWE1 protein expression among the five groups
cytotrophoblast, stromal cells, and endothelial cell are marked as S, C, (P,0.01). There was no significant difference among the larger
ST, and EN respectively. twin in discordant group, two concordant twin groups, and the
doi:10.1371/journal.pone.0033503.g002 singleton group (P.0.05).
Based on the Spearman correlation analysis, the HERVWE1
staining score was positively correlated with the HERVWE1
HERVWE1 and HCS transcript levels mRNA level (Rs = 0.229, P,0.05, GAPDH as control). Figure 3
In order to precisely investigate the HERVWE1 transcript levels, shows the correlation between the HERVWE1 staining score and
three internal reference genes were applied. The GAPDH and b- HERVWE1mRNA level. The HERVWE1 protein expression level
actin were commonly used housekeeping genes. The PCNA was an was consistent with the mRNA expression level in twins. Both of
index of cell proliferation which was used to eliminate the impact these measurements were increased in the smaller twin in
of cell proliferation on HERVWE1’s expression. As summarized in discordant group.

Table 3. HERVWE1 staining scores among the groups.

Larger twin in Smaller twin in Larger twin in Smaller twin in Singleton

discordant group (n = 10) discordant group (n = 10) concordant group (n = 10) concordant group (n = 10) group (n = 10)

HERVWE1 score 1.4360.33 1.7560.33{ 1.4360.35 1.4460.36 1.3260.40

Data are shown as the means 6 standard deviations.

{P,0.01 vs. other four groups.
doi:10.1371/journal.pone.0033503.t003

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 HERVWE1 Expression, Methylated Regulation in Twins

Figure 3. The correlation between the HERVWE1 staining score and the HERVWE1 mRNA level. The scatterplots showed the correlation
between the HERVWE1 transcript profile and the HERVWE1 staining score in all five groups. Using GAPDH as internal reference gene, the HERVWE1
transcript level was significant positively correlated with the HERVWE1 staining score (spearman correlation coefficient 0.229, P,0.05).
doi:10.1371/journal.pone.0033503.g003

The methylation profile of HERVWE1 transcriptional When the CG sites were sorted by methylation level, the order was
regulation region 3rd, 1st, 4th, 2nd and 5th for both discordant twin groups.
Pyrosequencing assay was applied in 21 pairs of discordant Spearman correlation analysis was applied to study the
twins and 10 singletons. The methylation levels of the 1st and 2nd correlation between HERVWE1 transcript level and methylation
CG sites and the average methylation levels of the five CG sites profile. While the GAPDH as internal reference gene, the
were lower in smaller discordant twin group compared with the HERVWE1 transcript level was negatively correlated with the
larger discordant twin group (P,0.01), and the singleton HERVWE1 promoter region the 1st(Rs = 20.434, P,0.01),
group(P,0.01). However, no differences were detected at the 2nd(Rs = 20.377, P,0.01),and mean(Rs = 20.510, P,0.01) meth-
3rd, 4th and 5th CG sites (P.0.05), as summarized in Figure 4. ylation levels (Figure 5). While the b-actin as internal reference

Figure 4. The methylation profile of the HERVWE1 promoter region, as determined by the pyrosequencing assay. There were 5 CG
sites in HERVWE1 promoter region. The pyrosequencing assay was applied to investigate the five CG sites methylation profile. The bar chart showed
the five CG sites methylation level. All of the CG sites methylation ratios were lower than 40%. The most hypermethylated site was the 3rd CG site.
The most hypomethylated site was the 5th CG site. The black, darkgrey, and lightgrey bars were represented the smaller discordant twin group,
larger discordant twin group, and singleton group respectively. The 1st, 2nd, and mean methlation level in smaller discordant twin group were lower
than that in larger discordant twin group (P,0.01) and singleton group (P,0.01).
doi:10.1371/journal.pone.0033503.g004

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 HERVWE1 Expression, Methylated Regulation in Twins

Figure 5. The correlation between HERVWE1 promoter region methylation level and HERVWE1 transcript levels. The scatterplots
showed the correlation between the HERVWE1 transcript profile and the HERVWE1 promoter region methylation level in discordant twin groups and
singleton group. Using GAPDH as internal reference gene, the HERVWE1 transcript level was significant negatively correlated with the HERVWE1

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 HERVWE1 Expression, Methylated Regulation in Twins

promoter region methylation level (A–C). Using b-actin as internal reference gene, the HERVWE1 transcript level was significant negatively correlated
with the HERVWE1 promoter region methylation level (D–F).
doi:10.1371/journal.pone.0033503.g005

gene, the HERVWE1 transcript level was also negatively correlated transcript level and the HERVWE1 promoter region methylation
with the HERVWE1 promoter region the 1st(Rs = 20.308, profile.
P,0.05), 2nd(Rs = 20.517, P,0.01),and mean(Rs = 20.428, We also compared the DNMTs transcriptional profiles among
P,0.01) methylation level (Figure 5). discordant twin group and singleton group. The results based on
GAPDH and b-actin as internal reference gene were consistent.
Global methylation (5-MC staining) There were no differences between the larger twin and the smaller
We used a semi-quantitative analysis method to score the slice twin in discordant group for DNMT1, DNMT3a, or DNMT3b
density. The staining was located in the nuclei of both of the (P.0.05). The smaller twin in discordant group expressed less
trophoblast and stroma cells. The positive nuclei were brown, and DNMT3b3 (P,0.05) and more DNMT3b7 (P,0.05) than the larger
the negative nuclei were blue after counterstaining with hema- twin in discordant group and singleton group. Figure 8 shows
toxylin. Comparing the 5-MC staining densities of the larger twin DNMTs transcriptional profiles based on GAPDH and b-actin as
in discordant group, the smaller twin in discordant group and the internal reference gene.
singleton group samples revealed no significant differences in
overall score among the three groups (P.0.05) (Table 4). Discussion
Figure 6 shows pictures of individual cases. For the smaller twins
in discordant group, we noticed the staining in the trophoblast Our data showed that HERVWE1 was more highly expressed in
cells (marked as S or C in Figure 6) was lighter than that seen in smaller twins than larger twins in discordant group. The
the stromal cells (marked as ST). However, the larger twin in HERVWE1 transcript level was negatively correlated with birth
discorant group and singleton group had no such distinction. weight. The methylation level of HERVWE1 promoter-region was
Therefore, we counted only the trophoblast cells using a semi- decreased in the smaller discordant twin group and increased in
quantitative analysis method, and we found that the smaller twin the larger discordant twin group, also well correlated with
in discordant group had a lower 5-MC staining intensity in HERVWE1 transcript level. We found that DNMT3b3 transcript
trophoblast cells than the larger twin in discordant group and the level was positively correlated with the HERVWE1 promoter
singleton group (P,0.01), but no differences between the larger region methylation profile. The DNMT3b3 mRNA level was
twin in discordant group and the singleton group downregulated, and the DNMT3b7 mRNA level was upregulated
(P.0.05)(Table 4). in the smaller discordant twin group.

DNMT transcript levels HERVWE1 expression in discordant twins

As indicated above, DNA methylation was closely related with Because previous studies by Ruebner and coworkers have
HERVWE1 transcript level. DNMTs are essential for establishing shown that syncytin-1 is decreased in IUGR, the increase in
and maintaining the cellular methylation patterns. The alteration HERVWE1 expression in smaller discordant twin placentas is
of DNMTs expression may attribute to the change of methylation slightly surprising [8,9]. However, it should be recognized that our
pattern. It may be interesting to explore the relationship between study population had unique features; the correlation between
the DNMTs transcript level and HERVWE1methylation profile in these characteristic features and the data has led to some
the placenta of discordant twins. We used real-time PCR assay to intriguing hypotheses, as discussed below.
analyze DNMTs transcriptional profiles among discordant twin Unlike previous studies using singleton placentas conforming to
group and singleton group. Thus we calculated the correlations the diagnostic criteria for IUGR, our study subjects were
between the DNMTs transcript levels and HERVWE1 methylation discordant (not IUGR) twins. All of our twins were in the normal
profile. While the GAPDH as internal reference gene, we only range for birth weight based on the twin-specific growth reference
found DNMT 3b3 transcript level was positively correlated with curve [26]. In our study, the boundary point for discordancy was
the HERVWE1 promoter region the 1st(Rs = 0.370, P,0.01), 20%, consistent with the majority of the literature. The average
2nd(Rs = 0.316, P,0.01),and mean(Rs = 0.485, P,0.01) methyla- weight discordance was 28.5%. Many researchers believe that
tion level (Figure 7). While the b-actin as internal reference gene, discordancy itself is not a risk factor for adverse neonatal outcomes
the result was also true. The DNMT3b3 transcript level was [12,27]. Twin weight discordancy and IUGR have different
positively correlated with the HERVWE1 promoter region the pathological significance, and they cannot be treated equally.
1st(Rs = 0.337, P,0.05) methylation level (Figure 7). However, Both the results from GAPDH and b-actin as housekeeping gene
there was no significant correlation between the other DNMTs supported the alteration of HERVWE1 transcript level in

Table 4. 5-MC staining scores for the discordant twin and singleton group.

Larger twin in discordant Smaller twin in discordant

group (n = 10) group (n = 10) Singleton group (n = 10)

Overall 5-MC score 1.760.4 1.660.4 1.760.4

5-MC in trophoblast cells 1.760.4 1.260.3*{ 1.660.5

Data are shown as the mean scores 6 standard deviations.

*P,0.01 vs. larger twin in discordant group;
{P,0.01 vs. singleton group.
doi:10.1371/journal.pone.0033503.t004

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 HERVWE1 Expression, Methylated Regulation in Twins

our study further verified the functional effect of HERVWE1 gene

on placenta.
Compensation to hypoxia is one of the characteristics of
placenta development. Hypoxia can induce VEGF expression and
trophoblast invasion in early trimester [29,30].Hypoxia may also
induce ROS production, which is one of features of IUGR and
preeclampsia [31], if hypoxia is not corrected. Decreased syncytin-
1 expression detected in the placenta of IUGR and the
preeclampsia cases seems to be a result of persistent hypoxia.
However, increased syncytin-1 expression detected in the smaller
discordant twin group may indicate compensatory reactions were
involved. The alteration may represent an early stage of IUGR.
Increased syncytin-1 may be part of the protective response to
hypoxia.
We also found that the larger discordant twin group’s
HERVWE1 expression was similar to that of the normal singleton
and concordant twin control group, whereas the expression in the
smaller discordant twin group was significantly higher than that of
the normal singleton group and concordant twin group. If
placental function is in the normal range, the placenta of the
smaller discordant infant will enlist more compensatory mecha-
nisms, such as release of syncytin-1, to make up for the growth
discrepancy. The larger infant’s situation is better, and as such, the
compensatory mechanism is not as distinct.
The increased HCS expression seen in the placentas of the
smaller discordant twin group supports our hypothesis. HCS, as
well as HCG, was highly expressed in the syncytium when
cytotrophoblast cells merged into syncytiotrophoblast cells. Its
expression status indicates placental function [32]. It is well known
that HCS levels significantly decrease during IUGR and
preeclampsia. Attempts have been made by some investigators
to use HCS or HCG as a marker of placental function and an
indicator of IUGR severity. The ß-HCG protein level was
significantly decreased in cytotrophoblast cells fractionated from
Figure 6. 5-MC staining in placentas from discordant twins and IUGR placentas in Ruebner’s study. In our study, the expression
singletons. In eukaryotic genomes, DNA methylation always occurs on level of HCS was consistent with HERVWE1 expression in each
a cytosine base, the fifth carbon of which is linked to a methyl group (- group. Moreover, the HCS transcript levels in both the larger and
CH3). IHC staining with an anti-5-MC antibody can quantitatively detect smaller discordant twin groups were greater than the normal level,
the intra-nuclear 5-MC density and intensity. A–H show placental tissues
with anti-5-MC IHC staining. The slices were incubated in a primary
as indicated by the singleton and twin control group. This finding
sheep polyclonal anti-5-methyl cytosine IgG antibody (diluted 1:200) indicates that not only did the placental function remain largely
overnight. A goat anti-sheep secondary antibody (diluted 1:400) was intact in both of our discordant twin groups but the alteration of
applied sequentially. The slices were developed with an immunoper- HERVWE1 expression was also related to the placental compen-
oxidase system. 5-MC was mainly detected in the nucleus. Positive cells sation capability. Although one of the infants was smaller than the
were stained brown. Negative cells were stained blue after counter- other, the placenta still had the capacity to promote its growth in
staining with hematoxylin. (A) The larger infant in discordant group
(magnification 10620). (B) The larger infant in discordant group order for it to catch up to the larger one. This compensatory
(magnification 10640). (C) The smaller infant in discordant group capability was very different from the decompensated changes
(magnification 10620). (D) The smaller infant in discordant group seen in the placenta during IUGR.
(magnification 10640). (E) A singleton infant (magnification 10620). (F) We applied the analysis of covariance to evaluate the influence
A singleton infant (magnification 10640). (G) The negative control of gestational age, since some researchers have found that
(magnification 10620). This singleton tissue slice was stained with 1%
throughout the entire pregnancy period, HERVWE1 expression
gelatin instead of the anti-5-MC antibody. (H) The negative control
(magnification 10640). Syncytiotrophoblast and cytotrophoblast cells increased as the gestational week increased in singleton pregnancy.
are marked as S and C respectively. Stromal cells are marked as ST. In the 3rd trimester, HERVWE1 mRNA expression reaches its
doi:10.1371/journal.pone.0033503.g006 peak, then it drops since 37 wks [33,34]. This may explain the
result of ours were different from Ruebner’s study, whose average
gestational age of the IUGR group was 36+3 wks. We found that
discordant twins. However, after eliminate the contribution of cell gestational age didn’t have significant effect on HERVWE1
proliferation, adjusted by PCNA, an indicator of cell proliferation, transcript expression in each group (P.0.05). We still observed
there was no significant difference between discordant twins. We the increased HERVWE1 transcript level in smaller fetuses of
speculated that the alteration of HERVWE1 transcript level was discordant twin when the gestational age was treated as covariate.
related to the cell proliferation, since it has been found that one of Furthermore, we couldn’t find the correlation between the
HERVWE1’s important function is proliferation [3,28]. Previous HERVWE1 mRNA expression and the gestational age in each
study had verified that the HERVWE1 expression was synchronous group. Thus, the differential expression of HERVWE1 observed in
with the PCNA expression in endometrial carcinoma tissue [28]. the study may be an intrinsic alteration for specific mechanisms
This consistent trend between HERVWE1 and PCNA expression in rather than simple increased gestational age. However, the

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 HERVWE1 Expression, Methylated Regulation in Twins

Figure 7. The correlation between DNMT3b3 transcript levels and HERVWE1 promoter region methylation level. The scatterplots
showed the correlation between the HERVWE1 promoter region methylation level and the DNMTs transcript profile in discordant twin groups and
singleton group. Using GAPDH as internal reference gene, DNMT3b3 transcript level was positively correlated with the HERVWE1 promoter region
methylation level (A–C). Using b-actin as internal reference gene, DNMT3b3 transcript level was positively correlated with the HERVWE1 promoter
region the 1st CG site methylation level (D).
doi:10.1371/journal.pone.0033503.g007

HERVWE1 expression trend during the whole twin pregnancy One of our study’s merits is that we have found evidence for a
season is still unclear, and we need to enroll more twin cases in differential change in cases of growth discrepancy in twins. This is
future study. difficult to study in a singleton case. This novel observation has

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 HERVWE1 Expression, Methylated Regulation in Twins

Figure 8. DNMTs transcript levels among discordant twins and singleton group. The values shown indicated the relative quantitation of
the DNMT mRNAs, which were standardized to GAPDH (A) and b-actin (B), normalized to the singleton control group. The DNMT 3b3 mRNA
decreased and DNMT 3b7 mRNA increased in smaller discordant twin group compared with the larger discordant twin group and singleton group.
doi:10.1371/journal.pone.0033503.g008

enriched our understanding of the molecular mechanisms involved in regulating gene expression in the placenta during twin
in the pathogenesis of fetal growth disorders. development. A meaningful methylation difference definitely
existed between the monozygotic twins during the intrauterine
Methylation alteration in monozygotic twins stage and have critical influence on fetal or placental development.
Our next important finding is the identification of a particular
alteration in methylation in the HERVWE1 promoter region in DNMT regulation in discordant twins
discordant, monozygotic twins, with methylation being decreased DNMTs may act as key enzymes in a complicated methylation
in the smaller twins. Previous studies in discordant, monozygotic regulation mechanism. In present study, we found that methyla-
twins also found epigenetic difference between the discordant tion profile of HERVWE1 transcriptional regulation region was
phenotypes. However, most of these studies were limited to adult correlated with the alteration of one of the DNMT3b’s isoform,
disease, such as in schizophrenia and multiple sclerosis [23]. DNMT3b3, indicating that some factors may control HERVWE1
Limited study was conducted in fetal stage. Our study used a expression through the modulation of methyltransferase enzymes.
unique study population to explore intrauterine growth differenc- The DNMT3b gene has 7 variants, the expression of which
es. Only dichorionic twins were enrolled, thereby avoiding the depends on different post-transcription spliced patterns [35]. Due
effect of vascular anastomosis between placentas. We found that to the lack of an intact catalytic domain, both 3b3 and 3b7 have
methylation of the HERVWE1 promoter region was decreased in limited catalytic activity. Although some researchers have found
the smaller twins but without according changes in global level. It that DNMT3b variants have diverse expression profiles in cancer
indicated that locus-specific methylation played an important role tissues [36], the research on these variants and their function is

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 HERVWE1 Expression, Methylated Regulation in Twins

very limited [35,37,38]. DNMT3b3 is a more commonly expressed adjacent to an upstream regulatory element (URE) of composite
variant, but its catalytic activity is controversial. We found that origin. The URE contains a trophoblast-specific enhancer (TSE,
DNMT3b3 transcript levels were lower in the smaller discordant light gray), which confers a high level of expression and placental
twin group. This result was consistent with the lower methylation tropism. TSE and U3 are considered the key methylation and
levels in the smaller discordant twin group. Without a complete transcriptional regulation control regions. These two regions
catalytic domain, DNMT3b7 theoretically has no enzymatic together are approximately 346 bp in length. There are 7 CG sites
activity. Furthermore, it can competitively bind to the enzyme’s in all, two of which are in the TSE; the other five are in the 59LTR
binding site on DNA. This mechanism might explain our finding U3. The CAP transcription initiation site is located at the 59 end of
that DNMT3b7 levels increased in the hypomethylated smaller the R region. Taking the CAP transcription initiation site as the
discordant twin group. This result suggests that DNMT3b7 acts as zero point, the 7 CG sites are located at 2336 bp, 2307 bp,
a negative regulator of methylation. Overall, the DNMT3b 2246 bp, 2208 bp, 2192 bp, 264 bp, and 243 bp. Our
variants’ tissue expression patterns and functions remain largely methylation study was focused on the first five CG sites. The
unknown. Although our study showed an outline of the expression target CpG dinucleotides are marked with vertical bars and circles.
of DNMTs in the placentas of discordant twins, and its potential (TIF)
contributions to methylation regulation in HERVWE1, the
underneath mechanism is still unclear. Further experiments Appendix Figure S3 Pyrograms. Two pyrograms of the same
should focus on normal placental tissue or trophoblast cell lines sample. Because pyrosequencing can read through only 50–
to explore the relationship between these variants and their 100 bp for each accurate pyrosequencing primer, we used two
functions in fetal growth and placental development. pyrosequencing primers to analyze the five CpG sites. The first
pyrosequencing primer was used to read the following sequence:
Conclusion TTT TGG GGY GGG TTT TTT TTT TGG GAT GAG GGT
We used discordant, monozygotic, dichorionic twins to study AAA AYG TTT GGA GAT ATA GTA ATT ATT TTG. The
HERVWE1 expression in the placenta and explore its potential second pyrosequencing primer was used to read the following
impact on fetal growth. We found that HERVWE1 transcript sequence: TAG TTG GAT TTT TTA GGT YGA TTA AGA
expression was negatively correlated with infant birth weight in ATT TTT AAG TTT AGT TGG GAA GGT GAT TAY GTT
discordant twins. HERVWE1 expression was increased in smaller TAT TTT TAA ATA YGG GGT TTG TAA TTT AGT TTA
discordant twins to levels higher than those seen in normal-growth TAT TT. S3A shows the results using the first pyrosequencing
twins and singletons. CG-site methylation in the HERVWE1 primer. The read out sequence was TTTTGGGGYGGGTTTT-
promoter region maybe a regulatory mechanism for suppressing TTTTTTGGGATGAGGGTAAAAYGTTTGG. This was com-
its expression in the monozygotic placenta. DNMT3b3 transcript pletely consistent with the targeted sequence. For the first CG site,
was positively correlated with methylation status in the HERVWE1 the C/(C+T) peak ratio was 24%. This result indicates that 24% of
promoter region and its mechanism is worth to be investigated in CG sites were methylated overall. Similarly, for the second CG
future study. site, the methylated cytosine fraction was 3% in total. S3B shows
the results using the second pyrosequencing primer. The read out
Supporting Information sequence was TAGTTGGATTTTTTAGGTYGATTAAGAAT-
TTTTAAGTTTAGTTGGGAAGGTGATTAYGTTTATTTT-
Appendix Figure S1 Gene maps of zygosity identification TAAATAYGGGGTTT. For the three CG sites, the methylated
assay. There are 15 autosomal short tandem repeat loci and 1 cytosine levels were 28%, 22% and 18%, respectively.
gender locus. The name of each locus is marked above the allele. (TIF)
Multiple alleles are possible at each locus. Each wave presents one
detectable allele. The serial number of each allele is labeled under Appendix Table S1 Primer Sequences. The detailed primer
each wave. If all of the 16 loci are identical, then the two infants sequences for real-time PCR and pyrosequencing assay.
are recognized as monozygotic twins. If any of the genotypes of the (DOC)
16 loci are different, then the two infants are dizygotic twins. S1A
and S1B show two individual infants’ genotypes. They were Acknowledgments
confirmed as monozygotic twins with identical genotypes at each
We thank all of the laboratory staff at the Hoskins Center, Mercer
locus.
University, for their instruction and assistance.
(TIF)
Appendix Figure S2 A schematic diagram of the Author Contributions
HERVWE1 promoter region CG sites. The HERVWE1
Conceived and designed the experiments: QF SWJ. Performed the
gene is located at 7q21.2. This is an LTR (long terminal repeat)-
experiments: YG ZMH HYS MCL. Analyzed the data: YZ. Contributed
element-rich region. Each LTR includes U3 (white), R (dark grey), reagents/materials/analysis tools: ZLW YML LHH. Wrote the paper: YG
and U5 (black) regions, in that order. The HERVWE1 ZMH.
transcriptional regulatory element is in the 59LTR U3 region

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