Epigenetics and its role in male infertility
Rima Dada & Manoj Kumar & Rachel Jesudasan &
Jose Luis Fernández & Jaime Gosálvez & Ashok Agarwal
Received: 28 November 2011 /Accepted: 17 January 2012
#Springer Science+Business Media, LLC 2012
Abstract Male infertility is a common and complex prob-
lem affecting 1 in 20 men. Despite voluminous research in
this field, in many cases, the underlying causes are un-
known. Epigenetic factors play an important role in male
infertility and these have been studied extensively. Epige-
netic modifications control a number of processes within the
body, but this review will concentrate on male fertility and
the consequences of aberrant epigenetic regulation/modifi-
cation. Many recent studies have identified altered epige-
netic profiles in sperm from men with oligozoospermia and
oligoasthenoteratozoospermia. During gametogenesis and
germ cell maturation, germ cells undergo extensive epigenetic
patterns in the sperm and oocytes. Increasing evidence sug-
effects on epigenetic processes controlling implantation, pla-
centation and fetal growth. This review provides an overview
of the epigenetic processes (histone-to-protamine exchange
and epigenetic reprogramming post-fertilization), aberrant
epigenetic reprogramming and its association with fertility,
possible risks for ART techniques, testicular cancer and the
effect of environmental factors on the epigenetic processes.
Male infertility is a complex problem where not only the
genes, but also the epigenetic factors play a crucial role.
There is an enormous interest in one potential cause of male
infertility—the aberrant epigenetic reprogramming in male
germ cells that can lead to sperm abnormalities. A number
of studies have explored the causes of male infertility and
now there is sufficient information supporting the idea that
epigenetic changes contribute to male infertility.
What is epigenetics?
The term epigenetics refers to changes in the phenotype
reintroduced the term to explain that gene action and expres-
sion that give rise to the phenotype . Epigenetic changes
encompass an array of molecular modifications of DNA or
Capsule Epigenetic mechanisms regulate germ cell development and
differentiation. Sperm epigenome is critical for optimal embryogenesis.
Sperm from infertile men are prone to epigenetic instability and this
may lead to increased incidence of imprinting defects in children
conceived by advanced assisted reproductive procedures.
R. Dada:M. Kumar
Laboratory for Molecular Reproduction and Genetics,
Department of Anatomy, All India Institute of Medical Sciences,
New Delhi 110029, India
Centre for Cellular and Molecular Biology,
Hyderabad 500007, India
J. L. Fernández
Unidad de Genética,
Complejo Hospitalario Universitario A Coruña (INIBIC),
As Xubias 84,
15006 A Coruña, Spain
Unidad de Genética,
Departamento de Biología,
Universidad Autónoma de Madrid,
28049 Madrid, Spain
A. Agarwal (*)
Center for Reproductive Medicine,
Cleveland Clinic 9500 Euclid Avenue, Desk A19.1,
Cleveland, OH 44195, USA
J Assist Reprod Genet
histones that are intimately associated with DNA. DNAwraps
around histones to form nucleosomes. Nucleosomes are pack-
aged into a higher order of structures called chromatin; mod-
ifications in chromatin control gene-expression in a spatio-
temporal manner [3–6]. The genome-wide approach to study-
ing epigenetics is defined as epigenomics.
Epigenetic mechanism of gene regulation
Two major modifications that occur in chromatin are DNA
methylation and post-translational histone modifications [3,
4]. DNA methylation is a biochemical process which
involves addition of a methyl group to the 5′ position of
the cytosine pyrimidine ring typically occurring in a CpG
dinucleotide . DNA methylation occurs as a result of
DNA methyltransferase (DNMT) activity. There are 3 main
DNMTs: i) DNMT1 —which plays a key role in main-
tenance of methylation; ii) DNMT 3a and iii) 3b, which are
de novo methyltransferases that methylate the genomic
DNA during early embryonic development . The changes
are acquired in a gradual rather than by an abrupt process [6,
9]. CpG islands are genomic regions that are approximately
500 base pairs long, which have a high frequency of CpG
sites (CG to GC ratio >55%) . These stretches of DNA
are located within the promoter region of about 40% of
mammalian genes which, when methylated, cause stable
heritable transcriptional silencing. Hypomethylation and
hypermethylation can occur simultaneously at different
regions in the genome .
Histones are basic proteins in eukaryotic nuclei, and they
package DNA into nucleosomes. H2A, H2B, H3 and H4
histones are integral part of nucleosomes. Histone modifi-
cations, such as acetylation, methylation, ubiquitylation and
phosphorylation, have emerged as the main players in epi-
genetic regulatory mechanisms. An intricate interplay exists
between modifications of the histone tails of H3 and H4,
some of which act antagonistically to regulate the conver-
sion from an active chromatin state to an inactive one
termed the histone code . Generally, the acetylation of
histones marks active, transcriptionally competent regions,
whereas hypoacetylated histones are found in transcription-
ally inactive euchromatic or heterochromatic regions. In
contrast, histone methylation can be a marker for both active
and inactive regions of chromatin. Methylation of lysine 9
on the N terminus of histone H3 (H3-K9) is a feature of
silent DNA and is globally distributed throughout hetero-
chromatic regions. On the other hand, methylation of lysine
4 of histone H3 (H3-K4) denotes activity and is found
predominantly at the promoters of active genes. H3-K9
methylation is a prerequisite for DNA methylation in fungi
and plants [13, 14]. DNA methylation can also trigger H3-
K9 methylation , as has been documented in mammals.
Epigenetic gene regulation during germ-cell
Epigenetic mechanisms regulate DNA accessibility through-
any stage of development. Each cell type has its own epige-
netic signature that reflects the developmental history and
environmental influences, and is ultimately reflected in the
phenotype ofthe cellandorganism. Atthe timeoffertilization
paternal genome delivered by the mature sperm has a haploid
genome and is packaged densely with protamines, whereas
maternal genome arrested at metaphase II is packaged with
histones. Upon fertilization, protamines are rapidly replaced
by histones and oocyte completes the second metaphase,
releasing the polar body. The H3 and H4 histones that associ-
ate with the paternal chromatin are more acetylated than those
present in the maternal chromatin [16, 17].
The epigenetic profile of germ cells changes dynamically
during development (Fig. 1) [18–21]. In post-implantation
mammalian embryos, pluripotent cells in the epiblast give
rise to primordial germ cells (PGCs). In females PGCs are
arrested in the prophase of meiosis-I whereas in males they
enter mitotic arrest. Germ cells undergo several changes in
their epigenetic profile during different stages of meiosis.
For example, premeiotic PGCs and spermatogonia, exhibit
unique patterns of histone modifications such as low
H3K9me2 levels [22–24] but in male germ cells, these
patterns change dynamically upon initiation of meiosis
. Changes in the composition and modification of his-
tones could contribute to chromatin modifications that are
required for proper meiosis, and for further maturation of
gametes . Both male and female germ cells undergo
final developmental changes after meiosis. In haploid round
spermatids, global nuclear remodeling occurs, although
some histone marks such as H3K9me2 on the inactive X
chromosome are retained [26, 27]. A testis-specific linker
histone variant H1T2 appears at this stage and plays a
crucial role in chromatin condensation during spermiogen-
esis . Later, a linker histone variant HIls1 (histone-1-like
protein in spermatids 1) is expressed in elongated sperma-
tids. In the histone-protamine exchange process, nuclear
histones become hyperacetylated during spermiogenesis
and shortly thereafter disassemble, replaced by transition
proteins (TP1 and TP2). At the final stage of spermiogene-
sis, transition proteins are removed and replaced by prot-
amines . The incorporation of protamines into sperm
chromatin induces DNA compaction, which is important for
the formation of spermatozoa and for providing a safe
environment for the genome. The presence of somatic-like
chromatin in the sperm nucleus could transmit different
epigenetic information to the offspring. Oakes et al. 
suggested that the genome-wide DNA methylation pattern
changes little during spermiogenesis, after the pachytene
J Assist Reprod Genet
spermatocyte stage. Histone methylation in spermatogenesis
is carried out by the H3-K4 and H3-K9 methyltransferase.
It has been reported that hyperacetylation of histone H4 is
associated with a histone-to-protamine exchange in haploid
spermatids [31–33]. Recently, Govin et al.  reported that
the double bromodomain-containing protein, BRDT (bro-
modomain testis specific) binds hyperacetylated histone H4
before accumulating in condensed chromatin and helps in
organizing the spermatozoon’s genome by mediating a gen-
eral histone acetylation-induced chromatin compaction and
maintaining a differential histone acetylation of specific
A factor named BORIS (brother of regulator of imprinted
sites), specifically expressed in male gonads, could be di-
rectly involved in the resetting of methylation marks during
male germ cell differentiation . The domains of BORIS
• Methylation of DNA
• Histone to protamine transition
• Histone removal and degradation
• Abnormal DNA methylation
• Altered expression of mRNAs
and other non-coding RNAs
while structure is
while structure is
• Phosphorylation of DNA (2 events)
• H2AZ and H3.3
• Double strand breaks
• Chromosomal nondisjunction
• Abnormal histone modification
• Protamine replacement errors
• Abnormal centrosome formation
• Apoptotic DNA fragmentation
(I & II)
(A & B)
Fig. 1 Epigenetic events
during spermatogenesis. In
primodial germ cells (mitosis),
DNA methylation occurs to set
up the paternal specific
imprints. Phosphorylation (in
meiotic cell) occurs to assist in
both recombination and XY
body formation. Ubiquitylation,
sumoylation and incorporation
of H2AZ and H3.3 variants are
all involved in XY body
occurs during spermiogenesis
to assist in the
Spermatocytogenesis can also
give rise to chromosome
non-disjunction during its
meiosis I and II along with
double strand breaks, abnormal
histone modification and
alteration in the expression on
mRNA and other non-coding
RNAs. DNA fragmentation is
the consequence of apoptosis
following double strand breaks
or abnormal protamination
J Assist Reprod Genet
have the same 11 Zinc Finger as CTCF (CCCTC binding
factor: a somatic regulator for expression of imprinted
genes), which binds to specific target DNA sequences and
plays an important role in the maintenance of differential
methylation patterns in somatic cells . CTCF is present
in both somatic and germ cells whereas BORIS is expressed
specifically in the male germ line. Studies [35, 37] have
shown that BORIS is linked with both methylases mediating
de novo methylation and demethylases mediating erasure of
Paternal impact on early embryogenesis
A growing body of evidence suggests that both genetic and
epigenetic abnormalities may contribute to idiopathic male
infertility, which may affect the outcome of in vitro fertiliza-
tion (IVF) . Advanced maternal age is one of the obvious
contributors to poor fecundity , but little is known about
is associated with decreased semen volume, sperm morphol-
ogy, and sperm motility, but no significant reduction in sperm
concentration . A number of studies have documented
age-dependent changes in the testis [41, 42].
High DNA fragmentation is associated with diminished
sperm count, motility, and morphology [42, 43]. Increased
DNA fragmentation also decreases fertilization and implan-
tation rates. The influence of sperm DNA methylation on
pregnancy was done on mice after using methylation deple-
tion by use of 5-aza-deoxycytidine. This molecule is base
analog that when incorporated into DNA decreases the level
of DNA methylation varying the gene expression . One
of the main problems of solving or throwing some light
about the actual role of DNA modifications on fertilization
is the absence of reliable techniques which allow easy and
reproducible analysis of the level of DNA modification in
each gamete. A simple Sperm Chromatin Dispersion (SCD)
based technology which offered stable results can be used to
analyze the amount of methylated DNA residues and the
level of DNA damage in each sperm (Fig. 2). This technique
consisted of using MABs against 5-Met-Citosine on a par-
tially denatured DNA molecule once the SCD test was
performed. The methodology is mainly based on previous
results where the SCD and FISH were used to characterize
the level of DNA damage and the presence of aneuploidies
(CITA). Figure 2 shows a panel of some results where
different levels of DNA methylation could be simultaneous-
ly correlated with the level of sperm DNA in each sperm. In
some cases DNA fragmentation fully correlates with an
abnormal DNA molecule while in other cases this does not
occurs. This points to the fact that probably methylation and
its consequence are characteristically displayed by each
sperm although its variations shall be associated to certain
pathological cases. Interestingly, adaptation of this tech-
nique may allow semiquantitative assesment of the level of
methylation cell by cell.
Fig. 2 Sperm DNA
fragmentation using the SCD
test (Halosperm) (panel a),
combined with the
simultaneous visualization of
sperm methylation (panel b)
using anti-5metyl-C. Panel 2c
was produced after merging a
and b. Semiquantitative repre-
sentation of the differential lev-
el of sperm DNA methylation
using a surface plot (panel d)
J Assist Reprod Genet
Histones are considered the best candidates for the trans-
mission of epigenetic information because of their influence
on the modification of chromatin structure, and access of
transcriptional machinery to genes [44, 45]. The methyl-
transferases facilitate gene silencing by mono-, di- or trime-
thylation of lysine or arginine [3, 46]. It has yet to be
determined whether modified histones play a crucial role
in gene expression during early embryogenesis or if abnor-
mal histone modifications in the sperm are associated with
diminished embryo development. Alterations in methylation
patterns can effect biallelic expression or repression of
imprinted genes resulting in various pathologies . Im-
paired spermatogenesis has been associated with aberrant
H4 acetylation . H4 hyperacetylation was also observed
in infertile men exhibiting Sertoli cell only (SCO) syn-
drome. Sperm of patients with asthenozoospermia and ter-
atozoospermia have decreased levels of DNA methylation
. Recently, researchers have begun to look at the contri-
bution to early embryogenesis by spermatozoa beyond those
of genetic factors. There is evidence of epigenetic contribu-
tion to complex diseases.
Epigenetic changes important for male gametes
Gonadal sex determination and testis development occur
between embryonic days 12 and 15 (E12 to E15) in the rat
(after midgestation in the human) and are initiated by the
differentiation of precursor Sertoli cells in response to the
testis-determining factor SRY. Aggregation of the precursor
Sertoli cells, PGCs, and migrating mesonephros cells (pre-
cursor peritubular myoid cells) promotes testis morphogen-
esis and cord formation. The fetal testis contains steroid
receptors and is a target for endocrine hormones. The an-
drogen receptor (AR) and estrogen receptor-b (ERb) are
present in Sertoli cells as well as in precursor peritubular
myoid cells and germ cells at the time of cord formation
(E14). Although the testis does not produce steroids at this
stage of development, estrogens and androgens have the
ability to influence early testis cellular functions. Treatment
with endocrine disruptors vinclozolin and methoxychlor, at
a critical time during gonadal sex determination (E8 to E15
in the rat), promotes an adult testis phenotype with de-
creased spermatogenic capacity and male infertility. This
study shows that external factors can induce an epigenetic
transgenerational phenotype through an apparent reprog-
ramming of the male germ line . It is not clear whether
steroidal factors acting inappropriately at the time of gonad-
al sex determination reprogram the germ line epigenetically
(altered DNA methylation) to cause the transgenerational
transmission of an altered phenotype or genetic trait.
Many epigenetic modifiers, including DNA methyltrans-
ferases, histone-modification enzymes and their regulatory
proteins play essential roles in germ-cell development.
Some of these are specifically expressed in germ cells
whereas others are more widely expressed. The crucial roles
of germ-cell-specific genes such as Dnmt3L and Prdm9
were revealed by conventional knockout studies [51–53].
A recent report showed that there are numerous intra- and
inter-individual differences in DNA methylation in human
sperm samples  which could contribute to phenotypic
differences in the next generation. Furthermore, it has been
reported that sperm samples from oligospermic patients
often contain DNA-methylation defects at imprinted loci
Epigenetics and protamine abnormalities
Marques et al.  suggested an association between aber-
rant sperm epigenetic modifications and altered spermato-
genesis. During differentiation of the male gamete, the
genome undergoes major changes that not only affect the
DNA sequence and genetic information by homologous
recombination but also alter its nuclear structure and epige-
netic information. One of the challenging issues is to under-
stand how the specific nucleoprotamine/nucleohistone
structure of the sperm nucleus conveys epigenetic informa-
tion and how it controls early embryonic events.
Cho et al.  have shown that both protamines 1 and 2
are essential for sperm function, and the haploinsufficiency
of either protamine 1 (P1) or protamine 2 (P2) results in a
reduced amount of the respective protein. The phosphoryla-
tion of protamines has also been shown to be very impor-
tant. Indeed, mutation of the calmodulin-dependent protein
kinase Camk4, which phosphorylates protamine 2, results in
defective spermiogenesis and male sterility [58, 59]. The P1/
P2 ratio in fertile men lies close to 1.0 [60–62] and ranges
from 0.8 to 1.2 [60, 61]. Perturbation of this ratio, in either
direction, is characterized by poor semen quality, increased
DNA damage, and decreased fertility [63–68].
An increasing amount of data now supports the hypoth-
esis that in mature mammalian spermatozoa, DNA is actu-
ally not homogeneously packed with protamines . Some
histones are retained in humans; in fact, the persistent his-
tones could be an important epigenetic code in the sperm
and may not be the result of inefficient protamine replace-
ment . Protamine replacement occurs in the elongating
spermatid stage of spermatogenesis (Fig. 3), long after the
completion of meiosis . The elongating spermatid also
undergoes other maturation events that affect its motility
and fertilization ability during the period of protamine re-
placement; but the events have not been linked to sperm
count. This has led to a hypothesis that the link between
abnormal protamine replacement and generally diminished
semen quality may be due to a defect in the unique system
J Assist Reprod Genet
of temporal uncoupling of transcription and translation dur-
ing spermatogenesis .
The study of two relatively novel areas—sperm epige-
netics and sperm transcriptome could be of particular inter-
est in men with protamine expression abnormalities.
Abnormal protamine incorporation into chromatin may af-
fect transcription of other genes. For example, in mice,
deregulation of protamines causes precocious chromatin
condensation, transcription arrest, and spermatogenic failure
. The human sperm nucleus, which retains 10–15% of
its original histone content, distributes it in a heterogeneous
manner within the genome . Various studies concluded
that histones that are retained bind specific regions, to con-
vey epigenetic information to the early embryo. If so, there
are obvious and profound implications for sperm with ab-
normal protamine replacement, and for the use of such
sperm, or any immature sperm, for intracytoplasmic sperm
injection (ICSI). Future research will classify and character-
ize the role of retained histones throughout the sperm
genome in mature sperm from fertile men as well as in
patients with known chromatin abnormalities.
Epigenetics and ART
In humans, the use of assisted reproductive technology
(ART) has been shown to induce epigenetic alterations
and to affect fetal growth and development. There is an
open debate about the influence ART on certain pathol-
ogies, especially those that are dependent on genomic
imprinting such as the Beckwith-Wiedemann or Angel-
man syndrome . Possible epigenetic risks linked to
ART techniques may result from either the use of sperm
with incomplete reprogramming or from in vitro embryo
procedures performed at a time of epigenetic reprogram-
ming [75–78]. Loss of gene imprinting may occur during
preimplantation under certain conditions of gamete han-
dling . Although epigenetic states are relatively sta-
ble, it has been estimated that the loss of epigenetic
control (epimutation) may be one or two orders of mag-
nitude greater than that of somatic DNA mutation .
Recent reports from various studies have shown that
epimutations not only lead to inappropriate expression
of the affected gene but may also expose hidden genetic
variation . It is possible that some sub-fertile couples
have a genetic predisposition to epigenetic instability,
which makes their offspring more susceptible to epige-
netic changes, independently of whether or not they are
conceived by ART. Epimutations affecting imprints can
arise during imprint erasure, imprint establishment or
imprint maintenance. It has been suggested by animal
model studies that loss-of-function mutations of DNA
methyltransferases affect all imprinted domains as well
as other chromosomal regions. For example, mutations of
Dnmt3a (de-novo methylase) and maintenance methylase
(Dnmt) could lead to loss of imprinting and embryonic
lethality . Another study has shown that targeted
disruption of Dnmt3L in mice caused azoospermia in
homozygous mutant males, and heterozygous progeny
of homozygous females died before midgestation. Anoth-
er study suggested that imprinting defects and subfertility
can have a common and possibly genetic cause and that
super-ovulation instead of ICSI may further increase the
risk factor of conceiving a child with an imprinting
defect. Based on this study and the study by Chang et
al.  it is tempting to speculate that super-ovulation
leads to the maturation of epigenetically imperfect
oocytes that would not have developed without treatment
and may disturb the process of DNA methylation in the
oocyte. It is therefore suggested that imprinting errors
can lead to spontaneous abortions. The influence of
sperm DNA methylation on pregnancy was documented
Fig. 3 Histone to protamine exchange during spermatogenesis
J Assist Reprod Genet
in one study where 5-aza-deoxycytidine (5-azaC) was
used to induce methylation depletion in mice. DNA
methylation level decreases when 5-azaC (a base analog)
is incorporated into DNA . It has been proposed that
the level of DNA methylation in human sperm could be
linked to their ability to initiate pregnancy by assisted
Epigenetics and testicular cancer
Testicular germ cells tumor (TGCT) represents approxi-
mately 98% of all testicular neoplasms and is the most
common malignancy among young males . Epigenetic
changes that deregulate gene expression are frequently ob-
served during the development of cancer. The epigenetic
equilibrium of the normal cell is disrupted during tumori-
genesis. In human neoplasms, at least two types of DNA
methylation defects are found: hypomethylation, character-
ized by a global loss of 5-methylcytosine, and hypermethy-
lation of regulatory regions of promoters, associated with
the silencing of tumor suppressor genes. Hypomethylation
was the first epigenetic abnormality to be identified in
cancer cells [87–89]. Studies in mouse models have indicat-
ed a causal relation between reduced levels of 5-
methylcytosine and tumor formation . In contrast to
the mere handful of oncogenes activated by DNA hypome-
thylation, a long list of tumor suppressor genes is transcrip-
tionally silenced by DNA hypermethylation in cancer cells.
TGCTs are believed to arise from primordial germ cells
(PGCs)—where DNA methylation and parental imprints
are erased and totipotency is restored [19, 91, 92]. A
genome-wide DNA methylation study using restriction
landmark genome scanning (RLGS) showed that the ge-
nome of seminomas was extensively hypomethylated and
virtually completely devoid of CpG island hypermethylation
. In contrast, the nonseminoma group was less exten-
sively hypomethylated and revealed variable CpG island
hypermethylation levels, which were comparable with
tumors of other tissues . From embryonic studies in
mice, a wave of demethylation immediately after fertiliza-
tion has been shown to erase the majority of methylation
marks in the genome, with the exception of some imprinted
genes and repeat sequences , leading to totipotency.
High expression of the de novo methyltransferases
DNMT3A and DNMT3B, as well as their homologue
DNMT3L, is significantly associated with the embryonal
carcinoma subtype . The presumptive testis-specific
chromatin regulator CTCFL (BORIS) and the pluripotency
marker POU5F1 (OCT3/4) have recently been proposed to
share properties with the cancer/testis associated genes in
being hypermethylated in somatic tissue and hypomethy-
lated in normal testis tissue [96, 97].
The effect of epigenetic sperm abnormalities on early
Imprinting errors in the developing fetus have been identi-
fied and shown to cause severe pathologies. Some studies
have also suggested that the use of ART increases the risk of
imprinting diseases. A study by Marques et al.  sug-
gested that an increase in abnormal methylation of the H19
gene in oligospermic men is associated with Beckwith-
Wiedemann syndrome. A decreased genome-wide methyla-
tion pattern in sperm has also been identified with poor
embryo quality in rats and decreased IVF pregnancy rates
in humans . Benchaib et al.  used 5-methyl-cytosine
immunostaining as an indicator of genome-wide methyla-
tion pattern in sperm. He showed that decreased global
methylation in semen samples from normospermic men is
related to a poor pregnancy outcome during IVF  sug-
gesting that global methylation status independently affects
embryogenesis. Mitchell et al.  found a correlation be-
tween the frequency of P1 transcripts and pregnancy rates in
men undergoing testicular sperm extraction (TESE) for
ICSI. This could suggest that epigenetic regulation of
DNA via nuclear packaging in the sperm is related to the
function of the mature sperm. Current knowledge of genetic
and epigenetic factors in sperm contributing to poor em-
bryogenesis is limited. Both the complex path of sperm
production and the delicate balance of epigenetic and genet-
ic factors during sperm maturation contribute to the forma-
tion of a mature sperm with the ability to fertilize an oocyte
and contribute to the developing embryo. A defect at any
step may manifest as male infertility.
Epigenetic regulation and nutrition
Epigenetic programming is tightly regulated, both tempo-
rally and spatially, during fetal development and lactation
[19, 20, 99]. Dietary supplementation with a methyl donor
during pregnancy increases the proportion of pups carrying
a methylated IAP (Intracisternal-A particles) sequence [100,
101]. There are many environmental and metabolic factors
that can influence patterns of histone acetylation and DNA
methylation, two major epigenetic marks. Metabolic factors
influencing these epigenetic modifications include intranu-
clear levels of acetyl-CoA for HAT activity, NAD+for Sir2
deacetylases, ATP for the deacetylation of chromatin sub-
strates by at least some HDACs and methyl donors of SAM
provided by the folate-methionine pathway. Once the spe-
cific epigenetic patterns corresponding to ‘labile’ and
‘locked’ situations are identified, these patterns could be
useful for diagnosis and prognosis . They may also
represent new types of targets for the development of novel
diets and drugs to prevent or to abolish aberrant gene
J Assist Reprod Genet
silencing, which may be associated with treatment failure
Most of these studies have looked at modifications of the
pattern of DNA methylation, as it is the easiest epigenetic
mark to study. Nutrition during early development can in-
fluence DNA methylation because one-carbon metabolism
is dependent on dietary methyl donors and on co-factors
such as methionine, choline, folic acid and vitamin B-12
. The availability of dietary methyl donors and cofac-
tors is therefore very critical during development. The epi-
genetic change, caused by a decrease in DNMT1 activity,
 can be prevented by folate supplementation .
Although not directly regulated by nutrition, maternal be-
havior also programs the epigenetic regulation (DNA meth-
ylation and histone acetylation) of the gluco-corticoid
receptor gene in the hippocampus and determines the stress
responses of the offspring [107, 108].
It was recently reported that a methyl-donor-deficient diet
in postnatal life can permanently affect the expression of
IGF2, resulting in growth retardation . This suggests
that the effects of nutrition are not only limited to the fetal
stage but nutrition during postnatal development can also
permanently alter the epigenetic regulation of imprinted
genes. In humans, diet has been shown to affect the DNA
methylation status of patients with hyperhomocysteinaemia.
This disease is characterized by the accumulation of S-
adenosylhomocysteine (an inhibitor of DNA methyltrans-
ferases). The impact of diet, nutrients or drugs on early
epigenetic programming must be seriously considered to
achieve a directed epigenetic regulation in spermatogenesis.
The role of environmental factors in epigenetic
Epigenetic modifications provide a putative link between
the environment and alterations in gene expression that
might lead to disease phenotype. An increasing body of
evidence from animal studies support the role of environ-
mental epigenetics in disease susceptibility. Environmental
exposures to nutritional, chemical and physical factors have
the potential to alter gene expression and therefore, modify
adult disease susceptibility in various ways through changes
in the epigenome. These genomic targets contain CpG
islands and other DNA sequences, although in some cases
the status of histone modifications in the same region,
determine levels of gene expression.
Monozygotic twins provide an interesting model for
studying the role of environmental factors in epigenetic
modifications . A large epigenetic study on monozy-
gotic twins  recently showed that twins are epigeneti-
cally concordant at birth in most cases, and that epigenetic
differences (DNA methylation and histone modifications)
accumulate with age in monozygotic twins. Remarkably, the
twins displaying the greatest epigenetic differences were
found to be those who had lived together for the smallest
amount of time. This finding underlines the relative impor-
tance of environmental factors in addition to intrinsic
Whether common diseases have an epigenetic basis is still
open to speculation, but if they do, this holds great promise
for medicine. Knowledge of genetic and epigenetic modifi-
cations of germ cells is necessary for the production of
functional gametes and for overcoming infertility. Categori-
zation of infertile men using a more detailed analysis of
DNA methylation patterns might reveal a new level of
reduced fertilization, implantation or pregnancy rates. Epi-
genetic studies offer an important window to understanding
the role of environmental interactions with the genome in
causing disease, and in modulating those interactions to
improve human health. Our increasing knowledge over last
10 years is beginning to be translated into new approaches
to molecular diagnosis and targeted treatments across the
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