EPIGENETICS IN REPRODUCTIVE MEDICINE

I.

Basic concepts of epigenetics

Despite the fact that every cell in a human body contains the same genetic material,
not every cell looks or behaves the same. Long nerve cells stretch out the entire
length of an arm or a leg; cells in the retina of the eye can sense light; immune cells
patrol the body for invaders to destroy. How does each cell retain its unique
properties when, in its DNA-containing nucleus, it has the same master set of genes
as every other cell? The answer is in the epigenetic regulation of the genes [1].
All cells in the human body carry the same DNA complement, which originates from
a single cell at conception. Highly orchestrated epigenetic mechanisms facilitate the
complex patterning required to ensure normal human development and support stable
regulation of appropriate patterns of gene expression in diverse cell types. Epigenetic
mechanisms define mitotically heritable differences in gene expression potential
without altering the primary DNA sequence. These mechanisms are highly regulated
by a large number of proteins that establish, read, and erase specific epigenetic
modifications, thereby defining where and when the transcriptional machinery can
access the primary DNA sequences to drive normal growth and differentiation in the
developing embryo and fetus. Several types of epigenetic marks work in concert to
drive appropriate gene expression. These include DNA methylation at CpG
dinucleotides, covalent modifications of histone proteins, ncRNAs, and other
complementary mechanisms controlling higher order chromatin organization within
the cell nucleus [2].
1. DNA methylation
DNAmethylation is typically associated with gene silencing through binding of
methylation-sensitive DNA binding proteins and/or by interacting with various
modifications of histone proteins that modulate access of gene promoters to
transcriptional machinery . In eukaryotic species, DNA methylation involves
transfer of a methyl group to the cytosine of the CpG dinucleotide .The vast
majority of mammalian DNA methylation occurs at CpG dinucleotides. CpGs are

distributed non randomly in the genome. They are concentrated in genomic

regions called CpG islands ranging in size from 200 bp to several kilobases.
These CpG islands, often unmethylated, are typically located within gene
promoters of actively transcribed housekeeping genes and tumor suppressor genes
[2]

Specific proteins, such as DNA methyltransferases, can establish or maintain

DNA

methylation

patterns.

Studies

in

mice

demonstrate

that

DNA

methyltransferases are essential for normal embryonic development. DNA

methylation is established de novo by the DNA methyltranferase (DNMT)
enzymes DNMT3a and DNMT3b
and maintained through mitosis primarily by the DNMT1 enzyme. DNMT1 is the
primary maintenance methyltransferase with a high affinity for hemimethylated
DNA. Its primary function is to copy the methylation patterns during replication.
DNMT1o, one developmental stage-specific isoform of DNMT1, is an oocytederived protein that enters nuclei at the eight-cell stage of early embryos and has
an essential role in maintenance of epigenetic marks [2].
DNA demethylation is also critical during primordial germ cell (PGC) and early
embryo development. This can occur via passive demethylation that is associated
with cell division or via active demethylation using excision repair mechanisms.
The active pathway requires hydroxylation of the 5-methylcytosine to 5hydroxymethylcytosine by the enzymes TET1 and TET2, followed by
deamination by AID and APOBEC1 before base or nucleotide excision repair. All
the enzymes in this pathway are expressed in mouse PGCs, suggesting a role in
gametic epigenetic reprogramming [2].
2. Histone Modification
The basic unit of chromatin consists of an octamer of histone proteins, two each
of H2A, H2B, H3, and H4. DNA wraps around this core, which provides
structural stability and the capacity to regulate gene expression (Fig. 1). Each core
histone within the nucleosome contains a globular domain and a highly dynamic
N-terminal tail extending from the globular domains (Fig. 1). Histone proteins
have tails that can have a number of post-translational modifications including

acetylation, methylation, phosphorylation, ubiquitylation, sumoylation, ADPribosylation, proline isomerization, citrullination, butyrylation, propionylation,
and glycosylation. Recently published data from the ENCODE Project
Consortium analyzed 11 such histone post-translational modifications including
acetylation and methylation, which mark active and repressive chromatin, as well
as modifications associated with transcription. Assessing various histone
modifications in a number of tissues, that data set identified different chromatin
states including inactive, bimodal, and active, each of which has different
functional properties. Bimodal states, in which a combination of active and
repressive marks are present in the chromatin of a promoter region of a gene,
facilitate rapid changes in gene expression, as might be expected during early
development, when differentiation and specification occur [2].

Chromatin assembly and disassembly are highly orchestrated processes that are
coordinated by histone chaperones and ATP dependent chromatin remodeling
complexes. Histone chaperones promote chromatin assembly by preventing nonspecific histone-DNA interactions while also promoting the correct histone-DNA
interactions [3].
3. Regulatory ncRNAs
Regulatory ncRNAs including small interfering RNAs (siRNAs), microRNAs
(miRNAs), and long ncRNAs (lncRNAs) play important roles in gene expression
regulation at several levels: transcription, mRNA degradation, splicing, and

translation. SiRNAs are double-stranded RNAs (dsRNA) that mediate posttranscriptional silencing, in part by inducing heterochromatin to recruit histone
deacetylase complexes [2].
MiRNAs comprise a novel class of endogenous, small (1824 nucleotides in
length); single-stranded RNAs generated from precursor RNA cleaved by two
RNA polymerase III enzymes DROSHA and DICER to produce mature miRNA.
These miRNAs can control gene expression by targeting specific mRNAs for
degradation and/or translational repression. They can also control gene expression
by recruiting chromatin-modifying complexes to DNA through binding to DNA
regulatory regions, thereby altering chromatin conformation (33, 34). Expression
of miRNA in human blastocysts correlates with maintenance of pluripotency in
embryo development [2].
LincRNAs, a subset of lncRNA, exhibit high conservation across different
species. They have been shown to guide chromatin-modifying complexes to
specific genomic loci, thereby participating in the establishment of cell type
specific epigenetic states. In embryonic development, expression of lncRNAs,
regulated by the pluripotent transcription factors OCT4 and NANOG, facilitates
cell lineagespecific gene expression (38). LincRNAs also play an important role
in developmental processes such as X-chromosome inactivation and genomic
imprinting [2].
II.

Epigenetics and Infertility

Factors Associated with the Infertility Phenotype
Several studies have identified different genetic and epigenetic factors as being
involved with the onset and progression of the infertility phenotype. However,
possible perturbed mechanisms involved in regulating gene expression in the disease
state are rather poorly understood. The classic nature versus nurture argument on
whether ones environment rather than genetic makeup could influence the onset of
infertility and other complex disorders has been reviewed by various studies [4].
Epigenetic Factors Influencing the Infertility Phenotype

Several histone deacetylases and demethylases have recently been identified and
attributed with regulation of chromatin state. However, their functional role and
association with diseased states is only just beginning to be understood, particularly
those factors affecting male and female infertility. Post translational modifications of
histone tails by factors including histone chaperones and methyltransferases are
involved in the proper regulation of gene expression. Due its dynamic nature and
plasticity, the landscape of chromatin can be altered, rendering a region of the
genome active or inactive. This altered state of packaging renders certain regions of
the genome more accessible to transcription machinery (euchromatin) and are marked
by DNA hypomethylation, RNA Pol II, and covalent histone modifications such as
histone 3 trimethylated at lysine 4 (H3K4me3) and the histone variant H2A.Z.
Inactive/repressed regions are known to be associated with DNA hypermethylation,
histone 3 trimethylated at lysine 27 (H3K27me3), and SUZ12 (part of the polycomb
group complex, PcG) [4].
Due to this, the main focus of recent epigenetic research has focussed on discovering
new factors involved in altering chromatin state and further looking at its involvement
in diseased and normal tissue. Recent studies have identified a critical role for the
JHMD2A (Jumonji C domain-containing histone demethylase 2A) histone
demthylase in male infertility, obesity, and spermatogenesis. Using knockout mice as
models, these studies identified a critical role for JHMD2A in the regulation and
expression of two genes, protamine 1 (OMIM #182880, Prm1) and transition nuclear
protein 1 (OMIM #190231, Tnp1) involved in the condensation and proper packaging
of chromatin in the male sperm. A higher degree of spermDNA compaction has
previously been attributed to the increased presence of highly basic protamine
proteins compared to histones in chromatin, a deficiency of which has been
associated with infertility in mice. Identification of other regulatory mechanisms
involved in the recruitment of factors, in addition to JHMD2A, involved in the
deposition of histones along with others affecting transcriptional activity of genes
involved in infertility will increase our understanding of mechanisms involved in both
perturbed and normal states [4].

a. Epigenetics and Female Infertility

Infertility
The World Health Organization defines the term primary infertility as the inability
to bear any children, whether this is the result of the inability to conceive a child,
or the inability to carry a child to full term after 12 months of unprotected sexual
intercourse. Primary infertility is sometimes known as primary sterility. However,
in many medical studies, the term primary infertility is only used to describe a
situation where a couple is not able to conceive [5].
Secondary infertility is defined as the inability to have a second child after a first
birth. Secondary infertility has shown to have a high geographical correlation with
primary infertility. Fecundity describes the ability to conceive after several years
of exposure to risk of pregnancy. Fecundity is often evaluated as the time
necessary for a couple to achieve pregnancy. The World Health Organization
recommends defining fecundity as the ability for a couple to conceive after two
years of attempting to become pregnant [5].
The terms infertility and infecundity are often confused. Fertility describes the
actual production of live offspring, while fecundity describes the ability to
produce live offspring. Fecundity cannot be directly measured, though it may be
assessed clinically. Typically, fecundity may be assessed by the time span between
a couples decision to attempt to conceive and a successful pregnancy [5].
Epigenetics
The epigenetic mechanism of action suggests that environmental factors alter how
a gene is expressed, but without directly changing the DNA sequence. Epigenetics
is the study of inherited changes in phenotype (factors that account for
appearance) that are not directly related to, nor explained by changes in our DNA
pattern. For this reason, this field of study is known as "epi," the greek root for
"above," indicating that a change has occurred that is not directly related to the
genetic code, but above it somehow. In epigenetics, non-genetic causes are
considered responsible for different expressions of phenotypes. Or, termed in a
different way, epigenetics describes changes in the expression of our genes that

are not caused by alterations in the DNA sequence. Essentially, a different factor
accounts for the change in gene expression [5].
Exogenous, or environmental components may affect gene regulation and thus,
potentially, subsequent expression in the phenotype. Changes to gene expression
induced by environmental contaminants can be permanent or transient. Research
has shown that epigenetic changes may in fact be reversed [5].
b. Epigenetics and male infertility
Aberrant epigenetic regulation, male infertility and embryonic development
It is crucial that proper regulation of epigenetic processes is maintained
throughout spermatogenesis to not only ensure proper sperm function, but also
proper embryonic development. It has been found that the sperm epigenetic
environment plays a role in establishing epigenetic marks in the embryo, thus
aberrant epigenetic regulation in spermatogenesis has a profound effect on both
male fertility and embryonic development [6].
1. DNA methylation
Improper DNA methylation of various genes has been implicated in abnormal
semen parameters, as well as several instances of male factor infertility. This
aberrant methylation can occur globally or be limited to one specific locus. A
study by Houshdaran et al. demonstrated that poor sperm concentration,
motility and morphology were associated with broad DNA hypermethylation
across a number of loci. Four of these sequences PAX8, NTF3, SFN and
HRAS were single copy sequences unique to non-imprinted genes.
Moreover, the repetitive element Satellite 2 was also found to be hypermethylated. The authors proposed that hypermethylation of these loci results from
the improper erasure of already established methylation marks rather than
aberrant de novo methylation following epigenetic reprogramming. The data
from this study suggest that methylation defects present outside of imprinted
loci may be a key factor in some cases of infertility. Recent research has
identified a critical role for the JHMD2A (Jumonji C-terminal containing
histone demethylase 2A) histone demethylase in male infertility, obesity and
spermatogenesis. Studies using knock-out mice models identified a critical
role for JHMD2A in the regulation and expression of protamine 1 and

transition nuclear protein 1, both of which are critical for DNA condensation
during chromosomal packaging in sperm. The lack of proper DNA packaging
in sperm has been associated with infertility in mice. It is possible that
aberrations of a number of other proteins regulating the activities of the
proteins involved in DNA compac-tion could cause infertility [6].
Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the folate
pathway that catalyzes the reduction of 5,10- methylenetetrahydrofolate to 5methylenetetrahydrofolate.

The

enzyme

maintains

bioavailability

of

methionine so it can be converted to S-adenosylmethionine, a methyl donor

for a variety of substrates including DNA. To better characterize the role of
MTHFR in spermatogenesis and male fertility, Khazamipour et al.compared
the methylation status of the MTHFR gene promoter in the testes of men with
non-obstructive azoospermia to men with obstructive azoospermia without
defects in spermatogenesis. It was found that 53% of men with nonobstructive azoospermia had hypermethylation of the MTHFR promoter,
whereas none of the men with obstructive azoospermia exhibited
hypermethylation of this region. These statistically significant data indicate
that MTHFR hypermethylation is a specific epigenetic aberration and may
strongly contribute to certain cases of male infertility. Interestingly, a study by
Kelly et al. demonstrated that adminis-tration of betaine during pregnancy,
nursing and post-weaning can indeed improve testicular histology and fertility
.Wu et al., in a very recent study concluded that hypermethylation of MTHFR
gene promoter in sperm was associated with idiopathic male infertility. The
authors demonstrated that the number of patients with hypermethylation was
three times to that of control individuals [6].
2. Genome imprinting
Appropriate establishment of genomic imprints is critical to the maintenance
of fertility. Indeed, both paternal and maternal imprinting defects have been
identified in several groups of men experiencing male factor infertility.
Substantial work has been directed towards confirming the presence of these
imprinting abnormalities. A study by Houshdaran et al. demonstrated that poor

semen parameters were linked with increased DNA methylation at several

differentially methylated loci, more than likely due to a defect in methylation
erasure during epigenetic reprogramming. These loci included PLAG1, a
maternally imprinted gene, DIRAS3 and MEST. An additional study by
Kobayashi et al. examined seven imprinted genes, including the paternally
imprinted GTL2 and H19 loci, in 97 infertile men. They found that 14.4% of
the patients studied exhibited abnormal paternal imprinting and 20.6%
exhibited abnormal maternal imprinting. Donors with normal sperm count and
motility were methylated at the H19 locus, whereas one patient with moderate
oligospermia and three patients with severe oligosper-mia showed no
methylation of the locus. Similarly, healthy donors displayed methylation at
the GTL2 locus while two patients with moderate oligospermia and four with
severe oligospermia displayed no methylation. Further sequencing of both loci
concurrently showed that one patient with severe oligospermia exhibited a
defect in both the H19 and GTL2 loci. Moreover, five of the ten patients with
severe oligospermia and three of the eight patients with moderate
oligospermia displayed aberrant methylation of maternally imprinted loci and
differentially
methylated regions [6].
Poplinski et al. compared the methylation status of the H19/ IGF2 imprinting
control region 1 (ICR1) and MEST loci in 33 healthy donors and 148 men
with idiopathic infertility. Normozoospermic men displayed high methylation
at the H19/IGF2 ICR1 locus and low methylation at the MEST locus, while
low methylation of the H19/IGF2 ICR1locus and high methylation of the
MEST locus was found to be associated with low sperm concentration. The
H19/IGF2 ICR1locus of men with idiopathic infertility was 89.6% methylated
with less than 40 million sperm in comparison to 95.9% methylated in normal
donors. As methylation of this locus increased, sperm count increased linearly.
Moreover, decreased methylation of H19/IGF2 ICR1locus directly correlated
with decreased sperm motility. Data from two different studies by Marques et
al. also indicated that some oligozoospermic patients and secretory

azoospermic patients with hypospermatogenesis exhibit loss of methylation at

H19. Similarly, in 2010 Boissonnas et al. found that many patients with
teratozoospermia

and

oligoasthenoteratozoospermia

exhibited

hypomethylation at variable CpG islands at the H19 locus. Hypermethylation

at MEST was more strongly linked with poor sperm quality than
hypomethylation at H19/IGF2 ICR1. This hypermethylation of MEST was
observed in samples from infertile men with less than 40% sperm motility and
less than 5% normal sperm morphology. Men with idiopathic infertility
exhibited MEST methylation of 9.6% in comparison to 4.3% in controls.
Furthermore, increased methylation of MEST linearly correlated with
decreased sperm motility. These data are supported by the findings of a study
by Marques et al [6].
3. Nuclear protein transitioning
The exchange of protamines for histones is a crucial step in the process of
spermatogenesis, causing the DNA to be tightly wrapped for efficient
transmission of nuclear material to the oocyte upon fertilization. It is known
that the Prm1 to Prm2 ratio (P1/P2 ratio) is strictly maintained and regulated.
Indeed, deviation from the standard ratio of 0.81.2 has been shown to lead to
infertility. A change in either direction of this ratio adversely affects semen
quality and DNA integrity. Patients with abnormally depressed or elevated
P1/P2 ratios are characterized by poor sperm concentration, motility and
morphology as well as decreased fertilization capabilities. Studies suggest that
the most common cause of infertility by aberrant protamine exchange is an
increase in the P1/P2 ratio caused by a decrease in Prm2 levels, although
improper regulation of Prm1 levels has also been implicated in some cases of
male infertility. Furthermore, a study by de Yebra et al. showed that men with
a higher P1/P2 ratio were also more likely to have lower total protamine levels
and higher intermediate protein levels. Moreover, this same study additionally
demonstrated that some infertile men completely lack Prm2 in their sperm
nuclei. There have been no cases reported of fertile men with severely altered
P1/P2 ratios; indeed, it appears to be a characteristic limited exclusively to

certain infertile males. Interestingly, a link between abnormal protamine

incorporation and aberrant genomic imprinting has recently been discovered.
Hammoud et al. found that infertile males with abnormal protamines exhibited
statistically significant hypermethylation at the imprinted loci KCNQ1, LIT1
and SNRPN. Moreover, this study also demonstrated that these patients
showed hypomethylation of the H19 locus [6].
As discussed previously, hyperacetylation of histone H4 is required in the
transition from histones to protamines. This step decreases the affinity of the
interaction between the sperm histones and DNA to allow the exchange for
transition proteins to occur. A study by Sonnack et al. showed that men
exhibiting qualitative and/or quantitative infertility have significantly
decreased levels of histone H4 acetylation associated with impaired
spermatogenesis. In the seminiferous tubules of men with round spermatid
maturation

arrest,

only

approximately

60%

of

spermatids

were

immunopositive for this hyperacetylation and many were multinucleated.

Moreover, infertile men with
qualitatively normal spermatogenesis

exhibited

approximately

90%

immunopositive spermatids and infertile men with qualitatively abnormal

spermatogenesis exhibited approximately 75% immunopositive spermatids.
This contrasts significantly with the almost all hyperacetylated round
spermatids in fertile men. Interestingly, spermatocytes in the seminiferous
tubules of men with round spermatid maturation arrest exhibited an additional
signal, indicating that early hyperacetylation of histone H4 may lead to
premature nuclear protein transitioning and subsequent infertility [6].
4. Chromosome structure
The global structure of chromatin is known to affect gene expression by
modulating which regions are available to be accessed by transcription factors
and other transcriptional proteins and which are not. This feature of epigenetic
regulation becomes particularly important when considering normally
expressed genes that are crucial for proper spermatogenesis and subsequent
oocyte fertilization. It has now been established that an increase in total

heterochromatic variants is strongly linked to some cases of male factor

infertility. Indeed, it has been demonstrated that there is an increase in the
frequency of chromosomal variants, from 32.55% to 58.68%, in infertile men
compared to controls. The large polymorphic variation 9hq+, located in
centromeric heterochromatin on chromosome 9, was shown to increase in
frequency from 4.25% in controls to 14.69% in men with severe infertility. It
is thought that this rise in heterochromatic regions, not only on chromosome 9
but amongst many chromosomes, may down-regulate normally active genes.
Indeed, polymorphic variations on the Y chromosome have been implicated in
male infertility based on this reasoning. All the major genes/loci known to be
epigenetically different in some infertile individuals are listed in Table 2 [6].

III.

Epigenetics and PCOS

1. miRNA
Little is known about the roles of miRNAs during follicular development,
steroidogenesis and in PCOS. Several studies on miRNA expression have been
done on intact ovaries (chicken, mice, pig, sheep and cattle), as well on the
different ovarian components, such as granulosa cells (mice, pig, horses and
human), theca cells, follicular fluid (humans, cattle and mares), cumulus cells
(mare), cumulus-oocyte complexes (COCs) (cattle) and corpora lutea (cattle) [7].
The possible modes of action for miRNA within the pathophysiology of PCOS
have only been sparsely investigated, and thus far, only a few miRNA-PCOS
studies exist (see Table 1).
It might be possible that identified PCOS susceptibility genes, such as
DENND1A, which interestingly also encodes miR-601, could result in genetic
and epigenetic factors overlapping, thus influencing miRNA target specificity. A

pilot study investigating global methylation in twenty PCOS women and 20 BMIand age-matched controls, using peripheral leukocyte DNA, showed no
significant differences in the median global DNA methylation percentages.
Despite the negative result, epigenetics may still play a part in PCOS
pathogenesis, since GC and ovary gene-expression could be tissue specifically
epigenetically modified in PCOS. Thus, PCOS is genetically complex with a large
degree of heterogeneity and is considerably influenced by environmental and
genetic cues, one of these being microRNAs [7].

2. Serum/Plasma miRNA Biomarkers for PCOS

Present abundantly in serum, miRNAs could serve as a non-invasive biomarker
for PCOS, as they have been shown to be stable in serum, are resistant to nuclease
activity and are easy to detect. It is not known specifically how miRNAs enter
serum or whether the miRNAs present in serum are disease-specific, since serum
is a result of different components secreted by various tissues and cells, and
identifying their cellular origin can be difficult. Currently, several other
biomarkers in the serum of PCOS women are used for diagnostic purposes, e.g.,
luteinizing hormone (LH) and androgen concentrations, as well as folliclestimulating hormone (FSH) [7].
A recent case-control study investigating 12 PCOS patients, 12 healthy females
and 12 male controls, subdivided further based on BMI levels, revealed that
obesity significantly reduced the expression of four miRNAs selected for
evaluation in whole blood: miR-21, miR-27b, miR-103 and miR-155 in control
women and men, but tending to show an increase in expression in PCOS women.
Further analysis of their hormone profile showed a positive correlation between

serum free testosterone levels and miR-21, miR-27b and miR-155. Perhaps, the
elevated free testosterone found in the PCOS samples could partly explain the
observed increase of these miRNAs. Further, bioinformatics analysis and target
gene analysis revealed that miR-21, miR-27b, miR-103 and miR-155 could be
involved in hormone metabolism, as well as reproductive cellular processes [7].
Using miRNA arrays, the expression of serum miRNAs in patients with PCOS
compared to age-matched controls has been evaluated . Following an initial
miRNA profiling based on a relative two-fold change in expression levels, nine
miRNAs (miR-222, miR-16, miR-19a, miR-106b, miR-30c, miR-146a, miR-24,
miR-186 and miR-320) were chosen for further analysis. The expression levels
for eight of the miRNAs were upregulated in serum from PCOS patients, whereas
miR-320 displayed decreased expression in the PCOS subjects. However,
following Q-PCR validation of the nine miRNAs expression in the entire study
population (n = 68 PCOS, n = 68 controls), only miR-222, miR-146a and miR30c remained significantly increased in the PCOS patients. Sensitivity and
specificity analysis, using receiver operating characteristic (ROC) curves and area
under the curve (AUC), revealed that a combination of the three miRNAs was
able to distinguish between the PCOS and controls. In addition, correlation
analysis adjusted for age and BMI showed that miR-222 strongly correlated
positively with serum insulin levels in PCOS women. Interestingly, upregulated
expression levels of miR-222 have also been associated with type 2 diabetes and
gestational diabetes mellitus. Further, miR-146a correlated negatively with serum
testosterone in PCOS women. Decreased miR-146 has been linked to
inflammation and insulin resistance in T2D individuals. An interesting
observation made by Long et al. was that most of the miRNAs present and
differentially expressed in ovarian tissue from PCOS women were not released
into the blood and, therefore, were not altered in PCOS serum [7].
In conclusion, identification of distinct miRNAs present within the circulation
would prove a useful tool for diagnosis and perhaps treatment of PCOS.
Comparing the miRNA profile of PCOS patients to healthy controls reveals that

miRNAs might contribute to the pathogenesis. Indeed, miR-21, miR-27b and

miR-103 are associated with PCOS, as well as metabolic features, such as obesity,
T2D, low-grade inflammation and adipogenesis dysfunction. Furthermore, insulin
sensitivity and the suppression of androgens have been associated with miR-222
and miR-146a, respectively. Profiling of serum miRNAs does not necessarily
reflect the more local changes within the ovary, and the functional role and
significance of miRNAs in blood from PCOS patients still need to be determined
[7]

3. MicroRNAs as Biomarkers for PCOS Based on Follicular Fluid Content

Comparing the miRNAs found in follicular fluid to the miRNAs found in the
blood
reveals the common occurrence of miR-186, miR-21, miR-155, miR-103, miR19a and miR-16, although with different expressions levels and significance
associated with PCOS. Moreover, the miRNA profile of follicular fluid varies
between studies, highlighting that PCOS is a complex and heterogenic syndrome.
Interestingly though, increased expression of miR-146 was found in serum from
PCOS patients and also in follicular fluid by Roth et al. and Sang et al.,
respectively. Adding to this, miR-222 and miR-24 were also found to be highly
expressed in follicular fluid, as well as identified in PCOS serum. A differential
expression, albeit in different directions, was observed for miR-320. Taken
together, this still warrants further studies [7].
4. Possible Role for miRNA in the Abnormal Follicular Development and Function
in PCOS
Many different theories have been brought forth in an attempt to explain the
mechanisms responsible for the impaired ovulation, abnormal follicular
development and excessive follicle formation commonly found in women with
PCOS, but with varying results. An altered appearance and function of granulosa
cells (GCs) with respect to FSH, LH and androgens has been proposed. Defects in
steroidogenesis by the theca cells (TCs) and increased activation of primordial
follicles, abnormal expression of anti-Mllerian hormone, increased follicle
survival and/or a decreased apoptosis rate have also been reported. Many of the

factors involved in these processes are still unknown and the mechanisms
unestablished [7].
Taken together, altered expression of ovarian miRNAs might play a role in the
processes determining the fate of granulosa cells (proliferation and differentiation
vs. apoptosis), and this might lead to the hyper-proliferating granulosa cells, as
seen in PCOS. The roles and mechanisms of miR-224, miR-320 and miR-383 in
GCs during folliculogenesis in general and in PCOS remain unknown [7].
IV.

Epigenetics and Endometriosis

1. DNA methylation
Recently it has been shown that DNMT1, DNMT3a and DNMT3b are over
expressed in endometriotic tissue. These findings are likely to provoke new ideas
regarding the origin and aetiology of endometriosis. For example, over-expression
of these enzymes would be expected to alter global DNA expression in
endometriotic cells. DNA microarray analysis of endometriotic tissue supports
this expectation since a substantial number of genes display significantly altered
expression patterns. Another possible explanation for these observed irregularities
in gene expression may be due to abnormalities in the regulation and function of
the major transcription factor, NF-B, in endometriosis (for review see Guo
2007 ). While this may be so, the introduction of epigenetics into the fray offers
new insights into the origin and progression of disease, such as the combined
effect of disrupting traditional and epigenetic regulators of transcription. In
support of this notion Wren et al demonstrated that epigenetic mechanisms such
as histone modifications, methylation and acetylation may play a role in the
aetiology of endometriosis. However, results from microarray studies provide an
unusual paradox. The majority of studies found almost the same number of down
regulated genes as there were up regulated in ectopic endometrial tissue. For
example, Kao et al reported 91 genes significantly over expressed and 115 under
expressed. Similarly Eyster et al 2002 and Eyster et al 2007 reported more genes
over expressed in ectopic endometrial tissue. Yet enhancement of DNMT function
in endometriosis should lead to increased levels of DNA methylation hence,

increasing the number of silenced or down regulated genes. This raises the
question as to how a system can be in place where global gene expression in
endometriotic cells should be down-regulated by over active methylation, and yet
the evidence clearly shows many genes are up-regulated. However, it should be
noted that Burney et al reported a higher frequency of under expressed genes in
the eutopic endometrium of women with endometriosis vs disease free controls. A
possible explanation to this paradox will be discussed in section 3.6. Of course it
must be considered that not all genes are epigenetically regulated, genetic
mechanisms are likely to play a significant role in aberrant gene expression in
endometriosis [8].
2. Epigenetic Modification of Steroid Synthesis and Receptors in Endometriosis
The deregulation of DNMTs is not the only evidence supporting the hypothesis
that epigenetics plays a major role in endometriosis. Izawa et al demonstrated that
the expression of the cytochrome p450 aromatase enzyme (CYP19) is dependent
on the methylation status of its promoter by treating endometriotic cells with the
demthylating agent 5-aza-deoxycytidine and observing the fold change in
aromatase mRNA expression. Current studies have reported either a weak or no
association between polymorphisms of the aromatase gene and endometriosis that
could account for the observed over expression of aromatase in endometriosis.
Those studies that have associated certain aromatase polymorphisms with
endometriosis have been criticised for faulty data analysis or non reproducible
results [8].
Aromatase is a key enzyme involved in the synthesis of estrogen and plays a
crucial role in the pathogenesis of endometriosis. With the exception of two
studies aromatase is reported to be highly up regulated in endometriotic cells
whilst being nearly undetectable in normal endometrium. The importance of
aromatase in the pathology of endometriosis is aptly demonstrated by the use of
aromatase suppressing drugs for the treatment of the disease. This class of drugs,
although with limited clinical data, have shown to be effective in the symptomatic
treatment of endometriosis. Aromatase is normally expressed in a cyclic fashion
throughout the menstrual cycle in eutopic endometrium however, expression

levels are consistently elevated in endometriotic cells. If the over-expression is

initiated by hypomethylation of the promoter

and maintained by aromatase

activating cytokines such as IL-6, IL-11 and TNF, all of which have been shown
to be dysregulated in endometriosis , a consequence would be over-expression of
aromatase leading to increased synthesis of estrone, which is converted to
estradiol, a potent estrogenic factor that initiates a number of pathways leading to
the proliferation and survival of endometriotic cells. The conversion of estrone to
estradiol is catalysed by the enzyme 17-hydroxysteroid dehydrogenase type 1
(17HSD I), which is reportedly up regulated in endometriosis. The increased
activation of aromatase in endometriotic cells leads to a self sustaining positive
feedback loop for estradiol production, whereby prostaglandin E2 (PGE2) activity
induces the up regulation of aromatase leading to increased estradiol levels. In
turn, this leads to the up regulation of the cycloxygenase-2 enzyme (COX-2)
resulting in the formation of more PGE2, an important factor in the pathology of
endometriosis thus the cycle becomes self perpetuating (Figure 25) [8].

Aberrant methylation of the aromatase promoter is not the only factor altering
gene expression due to epigenetic alterations in endometriosis. Regulation of
aromatase is mediated by steroidogenic factor-1 (SF-1) which is the aromatase
enhancer, and chicken ovalbumin upstream promoter transcription factor (COUP-

TF) which is its repressor. Indeed, SF-1 has recently been shown to be overexpressed in endometriotic cells. An explanation for the apparent over-expression
of SF-1 has been proposed by Xue et al who observed hypomethylation of the
CpG island near to its promoter region. The discovery of epigenetic modifications
in the promoters of aromatase and its enhancer SF-1 provide some understanding
of the establishment of the reported positive feedback loop. As with mutations,
once these epimutations are established, they are retained throughout each cellular
division, ensuring the survival of the endometriotic cells [8].
It is not only the synthesis of estrogen that is affected by aberrant methylation in
endometriosis. In order for estrogen to mediate its mitogenic effects within the
cell it must first bind to its receptor, of which there are two variants, estrogen
receptor (ERA) and estrogen receptor (ERB) coded for by separate genes.
Cells that over express estrogen receptors are highly sensitive to estrogenic
effects. Such cell types include breast and ovarian cancer cells which, as with
endometriotic cells, possess an enhanced proliferative capacity. Recent studies
have shown that the mRNA of one isoforms of the estrogen receptor (estrogen
receptor 2 gene, encoding estrogen receptor ) is over expressed in endometriosis.
This apparent over-expression was found to result from hypomethylation of the
CpG islands in the promoter of the ESR2 gene. ERB is important as it is known to
regulate several genes involved in signal transduction, cell cycle progression and
apoptosis, however in contrast to endometriosis several studies have shown ERB
to be down regulated in ovarian cancer and it thought that loss of ERB expression
may induce malignant transformation. It is also important to note that several
endocrine disrupters though to be risk factors for endometriosis mediate signalling
cascades via ERB. Therefore, not only does epigenetic modification lead to
enhanced estrogen production but it also leads to increased sensitivity towards
estrogen and estrogen-like compounds in endometriotic cells, resulting in a self
sustaining endometriotic cell population [8].
Due to abnormal estrogen synthesis and metabolism observed in endometriosis,
progestogenic agents are commonly administered to women with the disease in

order to suppress endometriotic cellular proliferation by down regulating estrogen

production and acting as an anti-inflammatory agent. The efficacy of
progestogens in relieving persistent pain symptoms associated with endometriosis
is relatively poor, and a reported 9% of women are completely unresponsive to
progestogen treatment. The relative inefficiency or total lack of response to
progestogenic treatment has thus, led some to conclude that endometriotic cells
are somehow resistant to the effects of progesterone. Evidence for this comes
from studies of the progesterone receptors in endometriotic cells. As with ER,
there are two progesterone receptor isoforms, PR-A and PR-B. Unlike the ER,
these encode as splice variants of the same gene and each has distinct functions
and distinct levels of expression in the eutopic endometrium, depending on the
phase of the menstrual cycle. PR-B is a transcriptional activator for several genes
containing a PR-B dependent promoter. PR-A, on the other hand is a
transcriptional repressor for PR-B and ER. Studies have shown that PR-B
expression is absent, and only very low levels of PR-A are expressed in
endometriotic cells, offering some explanation for progesterone resistance in
endometriosis. Additionally, Wu et al showed that the aberrant hypermethylation
of the PR-B promoter, reduces its expression to an almost silenced state. PR-A
and PR-B, although coded by the same gene, have distinct promoters, thus it may
be possible that aberrant methylation of the PR-A promoter may be responsible
for its reduced expression. Conversely Wu et al suggested that alteration of PR-A
expression may not be due to altered methylation of its promoter, but is likely due
to other as yet, unknown mechanisms. Nevertheless, it is reasonable to conclude
that there are epigenetic mechanisms by which estrogen production is both
enhanced and unopposed in endometriosis (Figure 26) [8].

3. HOXA10 in Endometriosis
HOX are a family of genes containing homeobox domains that act as transcription
factors essential for regulating genes associated embryonic development. Several
members of the HOX gene family play crucial roles during embryogenesis, for
example HOXA9, HOXA10, HOXA11 and HOXA13 are involved with the
development of the female reproductive tract, and unlike the majority of HOX
genes they are expressed into adulthood. The involvement of HOXA10 in the
development of the uterus affords specific interest with regards to endometriosis
since any aberrant expression of HOXA10 may result in abnormalities, in either
the function or morphology of the uterus. HOXA10 expression is reportedly down
regulated in patients with endometriosis, perhaps reflecting the findings that
patients with endometriosis are more likely to present with anatomical
complications of the reproductive tract. HOXA10 under-expression in
endometriosis patients may also explain the associated subfertility observed in
these patients since HOXA10 along with HOXA11 are responsible for successful
implantation of the embryo [8].

The origin of the down regulation of the HOXA10 gene in endometriosis was
investigated by Wu et al 2005 and Kim et al 2007. Wu et als study screened
eutopic endometrium of women with endometriosis, whereas Kim et al screened
eutopic endometrium from baboons with experimentally induced endometriosis.
Both studies identified hypermethylation in the promoter region of the HOXA10
gene. However, these studies examined only methylation patterns of HOXA10 in
the eutopic endometrium of endometriosis cases and controls, but did not examine
the methylation status of HOXA10 in ectopic endometrium. Wu et als study was
also confined only to women with stage III-IV endometriosis. Therefore, definite
conclusions regarding the aberrant expression of HOXA10 in endometriotic tissue
in humans cannot be made. However, a study by Lee et al 2009 using a murine
model of experimentally induced endometriosis, found inducing endometriosis
led to methylation dependant changes in HOXA10 expression in eutopic
endometrium. This essentially turns current thinking on its head, as it has long
been thought eutopic endometrium dictates the fate and function of ectopic
endometrium (via polyclonal origin of ectopic endometrium from refluxed
eutopic cells) not, as Lee et al demonstrated, the other way around. Although it
may be that interplay of signalling exists between the two cell types, the
mechanism by which ectopic endometrium can influence epigenetic alteration of
eutopic endometrium remains to be elucidated [8].
Interestingly, further study has reported that HOXA10 hypomethylation can be
induced by in-utero exposure to diethylstilbestrol (DES), a known endocrine

disruptor. The ramifications of DES exposure are discussed further in section 3.5.
The effect of altered HOXA10 expression in eutopic endometrium is obvious in
terms of uterine morphology and embryonic implantation. What the biological
significance of HOXA10 down regulation, in endometriotic tissue may be
remains speculative. Some clues may arise from microarray study of HOXA10
knockdown cells, which reported HOXA10 as a regulator of hundreds of genes
involved in a variety of cellular processes. Of particular interest was the finding
that HOXA10 knockdown led to a 5.78 fold increase in the CYP19 (aromatase)
gene, the significance of which is discussed in section 3.2. It is also important to
note that HOXA10 and HOXA11 are progesterone responsive genes and members
of the HOX family themselves regulate a number of other genes including
IGFBP-1 and integrins [8].
Nevertheless, the altered expression of HOXA10 in women with endometriosis
may provide an explanation for one of the most puzzling aspects of
endometriosis, which is, if retrograde menstruation is near universal, why do only
a fraction of women develop endometriosis? It may be that HOXA10 aberrations
result in improper development of the uterus. This is supported by the observed
uterine anatomical abnormalities in women with endometriosis such as increased
frequencies of septate uterus and the reported decreased elasticity of the
reproductive organs. Both aspects are thought to increase the volume of menstrual
reflux in some women, overwhelming the immune system which is thus unable to
remove all the refluxed endometrial cells [8].
4. Epigenetics and the Environment
Epigenetics provides a link between genotype and the environment, and how
exposures to different environmental, pharmacological and dietary elements can
translate into heritable changes in gene expression. During early mammalian
development the methylome is stripped and then re-applied in order to start
development from a blank state in which methylation errors are removed, for
this reason it was thought that alteration of epigenetic marks such as methylation
patterns could not effect subsequent generations. If epigenetic marks were
heritable then the complex phenotypic consequences they encode, which may

include disease phenotypes, must be heritable also. DNA methylation by DNMTs

is dependent on methyl donors such as S-adenosylmethionine (SAM), the major
methyl donor, which is synthesised as part of the methionine cycle. The formation
of this cycle is, in turn, dependent on dietary factors such as folic acid, vitamin
B12, choline and betaine. Animal studies have shown that restricting dietary
methyl donors produces a reduction in DNA methylation, and in some cases,
increased risk of developing tumours, indicating that epigenetic aberrations
mediated by dietary changes can result in complex disease phenotypes. The sparse
epidemiological studies which have reported on the influence of diet and
endometriosis suggest that a diet high in fruit and green vegetables and low in
meat and alcohol consumption is protective against developing endometriosis,
however some findings were inconsistent. From an epigenetic perspective a diet
high in fruit and green vegetables would provide a significant source of methyl
donors, which may protect against demethylation induced genetic instability
during foetal development. Alcohol consumption has been shown to alter histone
acetylation and methylation patterns which may account for the observed
estrogenic effect of alcohol [8].
V.

Epigenetics and Fibroids

1. DNA Methylation
Deoxyribonucleci acid methylation that occurs at the C5 position of cytosine,
resulting in 5-methylcytosine (5mC), mostly within CpG dinucleotides , is
involved in various developmental processes by silencing, switching, and
stabilizing genes. Deoxyribonucleci acid hypomethylation and imbalanced
expression of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) are
found in human uterine leiomyoma compared with the adjacent myometria. In
addition, the aberrant DNA hypomethylation is found in the distal promoter
region of ER-a (21188 to 2790) in the uterine leiomyoma compared with the
myometrium. A restriction landmark genomic scanning profile study found 29
aberrant methylation spots
(10 methylated and 19 demethylated) in leiomyoma compared with myometrium.
This study also found that DNMT1 and DNMT3A mRNA expression levels are
higher in leiomyoma compared with myometrium. Recently, Maekaya et al
identified 14 hypomethylated genes (FAM9A, CPXCR1, CXORF45, TAF1,

NXF5, VBP1, GABRE, DBX53, FHL1, BRCC3, DMD, GJB1, AP1S2, and
PCD11X) and one hypermethylated gene (HDAC8) located on the X chromosome
in uterine leiomyomas. Most recently, Navarro et al found 55 genes with
differential promoter methylation and concominant differences in mRNA
expression in uterine leiomyoma vs. normal myometrium. Eighty percent of the
identified genes showed an inverse relationship between DNA methylation status
and mRNA expression in uterine leiomyoma, and the majority of genes (62%)
displayed hypermethylation associated with gene silencing. Interestingly, they
found three known tumor suppressors genesKLF11, DLEC1, and KRT19
with
hypermethylation, mRNA repression, and protein expression in leiomyoma. These
results indicate a possible functional role of promoter DNA methylation-mediated
gene silencing in the pathogenesis of uterine leiomyoma [10].
2. Histone Modification
Histone modification is the second most important epigenetic factor that has a
critical role in regulation of gene expression. Histones proteins can be modified in
many ways in their N-terminal tail, including acetylation, phosphorylation,
methylation, ubiquitylation, sumoylation, and adenosine diphosphate (ADP)
ribosylation, deamination, and proline isomerization. Histone deacetylase 6
(HDAC6) is a regulatory factor in the endocrine traffic network and possesses
histone deacetylase activity and represses transcription. Wei et al examined the
HDAC6 expressions and its pathogenic role in the uterine leiomyoma. They found
a regular pattern of increasing HDAC6 and ER-a expression in leiomyoma
samples.
It is well known that epigenetic modifications are acquired during development
and play a central role in cellular differentiation and normal tissue and organ
function in adulthood. However, during crucial stages of development,
environmental exposures can alter genome state related to differentiation
programming of cells or organs, thus promoting disease susceptibility in later life.
A recent study reported that through nongenomic effects on developing uterus
during developmental reprogramming, environmental Es recruit the epigenetic

regulator EZH2 and reduce the levels of repressive histone mark H3K27me3 in
chromatin and promote uterine tumorigenesis [10].
3. MicroRNA
MicroRNAs are a novel class of small nonprotein-coding RNAs that regulate a
high number of biological processes by targeting mRNAs for cleavage or
translational repression. Studies have shown that several miRNAs, including let7,
miR-21, miR-93, miR-106b, and miR-200 and their predicted target genes, are
significantly dysregulated in uterine leiomyoma compared with normal
myometrium. Additionally, miRNA expression seems to be strongly associated
with tumor size and race. Pan et al reported that miR-21 is overexpressed in
leiomyomas, with specific elevation during the secretory phase of the menstrual
cycle in women who received depot-medroxyprogesterone acetate and oral
contraceptives, but decreased owing to gonadotropin releasing hormone agonists
(GnRHa) therapy. Recently Zavadil et al examined global correlation patterns
between altered miRNA expression and the predicted target genes in uterine
leiomyomas and matched myometria. They found that numbers of dysregulated
miRNAs are inversely correlated with their targets at the protein level. Patterns of
inverse association of miRNA with mRNA expression in uterine leiomyomas
revealed an involvement of multiple candidate pathways, including extensive
transcriptional reprogramming, cell proliferation control, mitogen-activated
protein kinase (MAPK), transforming growth factor (TGF)-b, WNT, Janus
kinase/signal transducers and activators of transcription signaling, remodeling of
cell adhesion, and cellcell and cellmatrix contacts. More recently, Fitzgerald et
al reported that elevated leiomyoma miR-21 levels are predicted to decrease
programmed cell death 4 (PDCD-4) levels, thus leiomyomas differ from other
tumors in which loss of PDCD-4 is associated with tumor progression [10].

Reference
1. Sarah C. P. Williams. Epigenetics. PNAS. 2013 February;110 (9): 3209

2. Michal Inbar-Feigenberg, M.D.,Sanaa Choufani, Ph.D, Darci T. Butcher, Ph.D., Maian

Roifman, M.D., and Rosanna Weksberg, M.D, Ph.D. Basic concepts of epigenetics.
Fertility and Sterility. 2013 March;99: 607
3. Varija N Budhavarapu, Myrriah Chavez and Jessica K Tyler. How is epigenetic
information maintained through DNA replication?. Epigenetics & Chromatin 2013, 6:32
4. SheroyMinocherhomji,1 Prochi F. Madon,2 and Firuza R. Parikh2. Review Article:
Epigenetic Regulatory Mechanisms Associated with Infertility. Hindawi Publishing
Corporation ,Obstetrics and Gynecology International Volume 2010, Article ID 198709, 7
pages doi:10.1155/2010/198709
5. FEMALE REPRODUCTIVE HEALTH AND THE ENVIRONMENT. Training Module
2 Children's Environmental Health.. Public Health and the Environment.World Health
Organization. www.who.int/ceh
6. Singh Rajender , Kelsey Avery , Ashok Agarwal , Review :Epigenetics, spermatogenesis
and male infertility. Elseveir: Mutation Research. 2011;727: 6271.
7. Anja Elaine Srensen , Marie Louise Wissing , Sofia Sal , Anne Lis Mikkelsen Englund
and Louise Torp Dalgaard. Review :MicroRNAs Related to Polycystic Ovary Syndrome
(PCOS). Genes 2014, 5, 684-708; doi:10.3390/genes5030684
8. Matthew David Rosser. The Emerging Role of Epigenetics in the Aetiology of
Endometriosis. De Monfort University Leiceister.
9. Serdar E. Bulun, M.D. Review Article: Mechanism Disease Uterine Fibroids.
N-Engl-J-Med-2013;369:1344-55. DOI:-10.1056/NEJMra1209993
10. Md Soriful Islam, Ph.D., Olga Protic, M.Sc., Piergiorgio Stortoni, M.D., Gianluca
Grechi, M.D. ,Pasquale Lamanna, M.D., Felice Petraglia, M.D., Mario Castellucci, M.D.,
Ph.D., and Pasquapina Ciarmela, Ph.D. Complex networks ofmultiple factors in the
pathogenesis of uterine leiomyoma. Original Articles : Gynecology and Menopause.