Epigenetic disorders and

male subfertility

line Chalas Boissonnas, M.D., Ph.D.,a Pierre Jouannet, M.D.,b and He
Ce
le
ne Jammes, Ph.D.c
a
HistologyEmbryologyBiology of Reproduction, CECOS, Ho ^ pital Cochin, AP-HP, Paris Descartes University, Paris, b CERSES
(UMR 8137)/CNRS/Paris Descartes University, Paris; and c INRA, UMR1198 Biology of Development and Reproduction, 78352
Jouy-en-Josas, France

Objective: To provide a link between epigenetics and male subfertility at the DNA, histone-protamine, and RNA levels and its
consequences on fertilization and embryo development.
Design: Review of the relevant literature.
Setting: University-based clinical and research laboratories.
Patient(s): Fertile and infertile men.
Intervention(s): None.
Main Outcome Measure(s): Critical review of the literature.
Result(s): Epigenetic markers can be modied in infertile patients. Epigenetic modications include methylation loss or gain on the
global level and on imprinted genes, high levels of histone retention in spermatozoa, and deciencies in some transcripts involved in
spermatogenesis. Interestingly, these abnormalities are all linked together, because DNA methylation maintenance depends on DNA
histone-protamine conguration which itself is stabilized by spermatozoal RNAs.
Conclusion(s): The paternal genome has long been considered to be silent and passive in em-
bryo formation. The epigenetic processes associated with the paternal DNA genome highlights
its importance in male fertility as well as for embryo development. (Fertil Steril 2013;99: Use your smartphone
62431. 2013 by American Society for Reproductive Medicine.) to scan this QR code
Key Words: Epigenetics, male infertility, methylation, histone, spermatozoal RNAs and connect to the
discussion forum for
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I
nfertility concerns 10%15% of cou- netic marks occurs in primordial germ tion, protamine-to-histone transition,
ples and is a growing concern of cells (PGCs). Second, male germ cell and histone modications that contrib-
modern society. Among infertility nuclei undergo a reorganization and ute to embryo genome activation and
causes, one-third are of male origin a condensation of their genome during embryonic development (9).
and one-third of combined infertility postmeiotic maturation, including Early evidence for a link between
(13). Genetic causes of male infertility DNA methylation, histone acetylation, epigenetic markers and male fertility
have been explored (4). Karyotypic implementation of histone variants, was demonstrated in mice by the
abnormalities account for
10% of and histone-to-protamine transition administration of a demethylating
male infertility (5), and microdeletion during spermiogenesis. Epigenetic sig- agent, 5-aza-20 -deoxycytidine. Reduced
of the Y chromosome (DAZ) is nals are crucial for the proper function- sperm production was observed in asso-
identied in 3% of azoospermic or ing of the sperm genome, and any ciation with reduction of testis and
severely oligozoospermic men (6). alteration during these processes may epididymal weight, reduced littermate
Although some specic mutations have contribute to altering sperm function size, and higher neonatal mortality
been identied, others responsible for and fertilization efciency. After fertil- (10, 11). However, it took several years
sperm defects remain unknown (7, 8). ization, the decondensation of the male before clearly demonstrating that each
The sperm epigenetic program is pronucleus follows an epigenetic re- level of epigenetic modications was
unique. First, a major erasure of epige- programming with DNA demethyla- linked to male fertility.
Received October 31, 2012; revised January 18, 2013; accepted January 21, 2013.
C.C.B. has nothing to disclose. P.J. has nothing to disclose. H.J. has nothing to disclose.
Supported by Assistance Publique-Ho ^ pitaux de Paris. THE DIFFERENT STEPS OF

line Chalas Boissonnas, M.D., Ph.D., Service dHistologieEmbryologieBiologie de
Reprint requests: Ce SPERMATOGENESIS
la Reproduction, CECOS, Ho ^ pital Cochin, AP-HP, Universite

Paris Descartes, 53 avenue de lObser-
vatoire, 75014 Paris, France (E-mail: celine.chalas@cch.aphp.fr). The mature spermatozoon is a highly
specialized cell with a agellum and
Fertility and Sterility Vol. 99, No. 3, March 1, 2013 0015-0282/$36.00
Copyright 2013 American Society for Reproductive Medicine, Published by Elsevier Inc.
a very compact nucleus. To achieve its
http://dx.doi.org/10.1016/j.fertnstert.2013.01.124 nal form, two main processes, meiosis

624 VOL. 99 NO. 3 / MARCH 1, 2013


and spermiogenesis, are required. During fetal life, germinal
stem cells occupy the seminiferous tubules of the testis, pro-
viding a pool of undifferentiated diploid cells called type A
spermatogonia. After birth in mice and at the onset of puberty
in humans, spermatogonia differentiate to type I spermato-
cytes which undergo two meiotic divisions to produce sper-
matids. Spermatids then go through a unique process, called
spermiogenesis, that enables a round cell, with nuclear tran-
scription and translation activities, to become a agellated
cell containing a compact nucleus without nuclear activity.
DNA compaction in male germ cells is evolutionarily a highly
conserved process (12). It is a fundamental biologic process
and a prerequisite for transmission of the male genome to
the next generation. Several studies suggest that this sperm-
specic genome packaging structure conveys an important
epigenetic message to the embryo (13, 14). In early
spermatids, DNA is compacted around nucleosomes, the
universal organization units of the genome. A nucleosome
is comprised of DNA coiled around an octamere of
canonical histones H2A, H2B, H3, and H4. The drastic
compaction of the male genome is characterized by the
replacement of a large number of histones by small basic
nonhistone proteins, rst by transition proteins (TNP1 and
TNP2) and nally by protamines (P1 and P2) (15).
Chromatin compaction occurs through the formation of
disulde bounds between protamines, producing toroidal
structures (16) and thereby creating a chromatin structure
620 times more compacted than histone-bound DNA
(17, 18). Once this transition is achieved, no more nuclear
activity is allowed and only transcripts inherited from early
spermatids, such as mRNAs and small interfering RNAs
(siRNAs), are present in the nucleus.
Considering the silent state of the compacted nucleus, it
has been thought for a long time that spermatozoon tran-
scripts did not play a role in embryo development and that
only maternal transcripts were involved. Recent advances
have demonstrated that the paternal nucleus conguration
and sperm transcripts contribute to early embryo develop-
ment (13, 14). Therefore, understanding the epigenetic
processes in the male germ cells may contribute to
understanding paternal effects on early embryonic
development (19, 20).
The aim of the present review is to focus on epigenetic
markers of male germ cells, such as DNA methylation and his-
tone variants and modications, on their interactions with
chromatin and with noncoding RNAs and to explain how
they interfere with male fertility. Finally, the potential conse-
quences of epigenetic alterations carried by male germ cells
on embryo development will be discussed.
METHYLATION AND MALE FERTILITY
DNA methylation, rst described in the 1970s (21), consists in
the addition of a methyl group from S-adenosyl-methionine
to the fth position of the cytosine ring (5meC) in CpG dinu-
cleotides: 3%5% of the cytosine residues in mammalian ge-
nomic DNA appear in the form of 5meC (22). The CpG islands
are dened algorithmically as sequences with an observed-to-
expected ratio of CpG of >0.6 and with a length of >500 bp
VOL. 99 NO. 3 / MARCH 1, 2013
Fertility and Sterility
(23). The distribution of CpG sequences in the mammalian ge-
nome is nonrandom. The strong CpG island promoters are
mostly unmethylated (such as housekeeping gene promoters).
In contrast, CpG islands located in the promoter and/or in reg-
ulatory regions of transposable elements are methylated, in-
hibiting the parasitic transposable and repetitive elements
from replicating. CpG-poor promoters are frequently hyper-
methylated in somatic cells. DNA methylation patterns at reg-
ulatory regions are usually stable, but dynamic de novo
methylation of regulatory regions can occur during develop-
ment to prevent their reexpression (24). Emerging data
indicate that DNA methylation may also occur in embryonic
cells at cytosine residues in CpA or CpT dinucleotides (25, 26).
Recently, Sasakis team reported accumulation and loss of
asymetric non-CpG methylation during male germ-cell de-
velopment. However, the biologic function of non-CpG meth-
ylation remains unknown (27).
The establishment of methylation in germ cells is a unique
process and is necessary for proper spermatogenesis and
sperm production (19). In mouse germ cells, it was well dem-
onstrated that there was a global demethylation-
remethylation process involving widespread erasure of
somatic-like patterns of DNA methylation followed by the es-
tablishment of sex-specic patterns by de novo DNA methyl-
ation. (22, 2831). Demethylation takes place in PGCs of the
developing embryo between 8.013.5 days postcoitum (dpc),
erasing all methylation markers (3234). A specic
remethylation begins at around 15.5 dpc (3537) (Fig. 1).
This erasure process specically concerns parental
imprinting. Parental imprinting is an epigenetic
phenomenon that mediates monoallelic parent-of-origin
specic expression of genes (38). Imprinting marks are
erased in PGCs and a specic remethylation occurs in
spermatogonia and type I spermatocytes, before their entry
into meiosis, so that all spermatozoa transmit the correct
paternal imprint (28). Most of the methylation is acquired
during fetal life but complete levels of DNA methylation are
not achieved until the pachytene spermatocyte stage, thus
postnatally and before meiosis (39). Only four imprinted
loci are methylated in the male germ line, namely Igf2/H19,
Rasgrf1, Dlk1-Gtl2, and Zdbf2 (40). DNA methylation de-
pends on a family of enzymes called DNA methyltransferases
(DNMTs). DNMT1 ensures methylation maintenance (41), and
DNMT3A, 3B, and 3L specically allow the methylation pro-
cess in germ cells (Table 1). DNMT3A and 3B have a catalytic
activity, whereas DNMT3L lacks this catalytic activity and
acts as a cofactor to DNMT3A (42, 43). Their activities are
absolutely essential to the normal completion of
spermatogenesis. Indeed, conditional Dnmt3a knockout
(KO) in germ cells impairs spermatogenesis by germ cell
apoptosis and is associated with methylation loss of
paternally imprinted genes (44). Dnmt3l KO induces meiotic
arrest in spermatocytes owing to chromosome asynapsis. A
deregulation of the methylation process of imprinted genes
and repeated sequences is also observed (45).
This process has been extensively studied in mice, in
which precise kinetics of erasure and remethylation are
known, but to date it has not been well described in humans.
Only two studies have reported methylation marks of
625
VIEWS AND REVIEWS

FIGURE 1

Summary of DNA methylation, chromatin modications, and sperm RNA regulation during spermatogenesis. A process of demethylation-
remethylation takes place in germ cells before the onset of meiosis. The methylation markers are then maintained until fertilization.
Modications of histones, such as methylations and acetylations, condition the good processing of spermatogenesis. Spermiogenesis is
characterized by histone-to-protamine transition, except for some histones specically conserved. Some sperm RNAs are retained in the nal
spermatozoa. These epigenetic markers interact so that the spermatozoa gets the correct epigenetic markers.
Boissonnas. Epigenetics and male fertility. Fertil Steril 2013.

imprinted genes in germ cells of adult men. Both studies con- proper erasure of methylation marks in cases of oligoastheno-
rmed the methylation of the H19 differentially methylated teratozoospermia (OAT) rather than from de novo methylation
region (DMR) in spermatogonia and the expected demethy- following epigenetic reprogramming. They also suggested that
lated state of MEST/PEG1 in spermatogonia and type I sper-
matocytes (46, 47). In humans, it is commonly considered
that, as in mice, methylation markers are acquired before TABLE 1
entry into meiosis. Nevertheless, it remains unknown if the
Functions of the main DNA modier enzymes and their timing of
process takes place during fetal life, in the perinatal period, action during spermatogenesis.
and/or at the onset of puberty.
Timing Enzyme DNA modication
Several studies have explored the relationship between
DNA methylation levels and male fertility in humans. Using Mitosis DNMT1 DNA methylation maintenance
DNMT3A DNA de novo methylation
immunostaining of DNA methylation, a rst study linked
DNMT3B DNA de novo methylation
high global methylation levels of ejaculated sperm DNA to DNMT3L DNMT3a cofactor
high pregnancy rates, associating methylation defects to infer- HAT H3 and H4 acetylation
tility (48). The Houshdaran et al. study, based on Methylight MII2 (HMT) H3K4 methylation
Meiosis HDAC H3 and H4 deacetylation
screening of 36 target genes with the use of the Illumina plat- LSD1/KDM1 (HDM) H3K4 demetylation
form, showed improperly elevated levels of DNA methylation HMT H3K9 and H3K27 methylation
of imprinted and nonimprinted genes and repetitive elements Spermiogenesis HAT H4 hyperacetylation
in poor-quality semen samples (49). The authors proposed JHDM2A (HDM) H3K9 demethylation
Boissonnas. Epigenetics and male fertility. Fertil Steril 2013.
that these elevated methylation levels resulted from an im-

626 VOL. 99 NO. 3 / MARCH 1, 2013


not only imprinted genes, but also broad epigenetic defects
were associated with sperm defects. Using the Illumina Inn-
ium Human Methylation 27 Beadchip assay, Aston et al. de-
scribed a genome-wide altered DNA methylation pattern in
men with altered semen parameters (50), linking global meth-
ylation levels of sperm with fertility. Considering the impor-
tance of imprint acquisition during spermatogenesis,
Marques et al. studied some imprinted genes in different sperm
populations (51, 52). They compared the paternally methylated
H19 DMR and unmethylated MEST DMR in spermatozoa from
fertile and infertile men. A loss of methylation of H19 DMR
was found in men with OAT, and an association between
H19 DMR methylation decrease and decreased sperm count
was observed (51, 52). An unexpected and abnormal
methylation state of MEST DMR was also observed in
oligozoospermic patient sperm (52). These ndings were
conrmed by a Japanese team on several other imprinted
genes showing loss of methylation at H19 and GTL2 DMRs
and improper methylation acquisition at PEG1, LIT1, ZAC,
PEG3, and SNRPN loci in cases of moderate to severe
oligozoospermia (53). An association between abnormal
imprinting in spermatozoa and spontaneous abortion after
ART was found with the same imprinting errors reported in
the sperm and in the abortion product (53, 54). However, in
those studies, methylation analysis was performed using the
bisulte conversion and cloning-sequencing technique. That
technique allows allele-specic methylation analysis but is
poorly representative of the methylation status of the studied
sample. Using bisulte conversion coupled with pyrosequenc-
ing, Boissonnas et al. performed a quantitative analysis of
methylation levels at each CpG position included in IGF2
and H19 DMRs (47 different CpGs) in a population of normo-
zoospermic and oligozoospermic men (55). They conrmed
that the drastic loss of methylation restricted to IGF2 DMR2
(primary IGF2 DMR) and H19 DMR correlated with the severity
of the oligozoospermia. These data may suggest that some
DMRs are more sensitive to methylation defects than others.
This could depend on the different DNA compaction state of
these sites (55). Methylation errors (hypermethylation or deme-
thylation) associated with OAT were next conrmed by other
teams using several techniques (50, 5658).
Finally, some groups studied the methylation state of some
promoter genes involved in the spermatogenesis process. Ab-
normal methylation of the DAZL promoter (59) and the
CREM promoter (60, 61) were reported in men with OAT.
More interestingly, the methylation status of the
methylenetetrahydrofolate reductase (MTHFR) gene promoter
was studied (62). This gene encodes a key enzyme of the
folate pathway, which maintains methionine bioavailability.
Methionine can be converted to S-adenosylmethionine, the
universal methyl donor for many substrates, including DNA.
A hypermethylation of the MTHFR promoter was found in
53% of nonobstructive azoospermia (63) and in some cases
of idiopathic infertility (64).
All of these studies support the hypothesis that
sperm DNA methylation patterns of imprinted as well as non-
imprinted genes are essential for normal sperm function,
fertility, and embryo development. However, the causes of
these methylation errors, as well as the timing of their occur-
VOL. 99 NO. 3 / MARCH 1, 2013
Fertility and Sterility
rence, remain unknown. Are these methylation errors ac-
quired during fetal or early postnatal development? Are
they linked to abnormal methylation maintenance during
spermatogenesis? It seems to be unlikely that altered expres-
sion of DNMT, particularly 3A or 3L, could be responsible for
these disorders, because anarchic methylation and demethy-
lation are observed in oligozoospermic men. A possible expla-
nation could be that methylation abnormalities are linked to
abnormal DNA conguration during spermiogenesis, where
histones and protamines play a role in the maintenance of
the methylation markers of male gametes (65).
HISTONE MODIFICATIONS AND MALE
FERTILITY
Histones are subjected to posttranslational modications on
histone tails including phosphorylation, methylation, acety-
lation, and ubiquitination on different residues. Each of these
modications works alone or in concert, under the name of
the histone code, to inuence activation or inhibition of
gene transcription (66). Activation of genes is commonly as-
sociated with acetylation taking place on the lysine residue
(K), particularly on H3 and H4. The establishment and re-
moval of aceylation is accomplished by histone acetyl trans-
ferases (HATs) and deacetylases (HDACs), respectively (66)
(Table 1). Histone acetylation relaxes chromatin and makes
it accessible to transcription factors, whereas deacetylation
is associated with gene silencing (66). Acetylation of H3
and H4 lysine residues is high in male stem cells and is re-
moved during meiosis, but the reacetylation of H4K in elon-
gating spermatids is a prerequisite for histone-to-protamine
exchange (67) (Fig. 1). The use of an HDAC inhibitor, trichos-
tatine A, resulted in a signicant reduction of the number of
spermatids and severe male infertility (68, 69). Methylation of
H3 or H4 lysine residues can promote gene activation or
repression and is regulated by histone methyltransferase
(HMT) and demethylase (HDM) families (70) (Table 1). H3K4
methylation is generally associated with gene expression,
whereas H3K9 and H3K27 methylation is linked to gene
silencing and heterochromatin. The timing of establishment
and removal of methylation markers is critical to
spermatogenesis: Mono-, di-, and trimethylation
modications of H3K4, H3K9, and H3K27 are essential for
the progression through spermatogenesis (71, 72). For
example, the methylation level of H3K4 peaks in
spermatogonial stem cells and is required for stem cells to
begin differentiation and to go on to become spermatocytes.
It then diminishes during meiosis. In contrast, the
methylation level of H3K9 and H3K27 increases during
meiosis, but the removal of H3K9me at the end of meiosis is
essential to the onset of spermiogenesis. Specic enzymes
play a role in orchestrating these modications. In mice, the
reduction of the histone H3K4 methyltransferase MII2
activity resulted in a dramatic decrease of the number of
spermatocytes by an apoptotic process, inducing
a developmental block in spermatogenic differentiation (73).
The loss of LSD1/KDM1, an H3K4 HDM, during meiosis
resulted in germ cell apoptosis and sterility (74, 75)
(Table 1). Targeted disruption of JHDM2A in mice resulted
627
VIEWS AND REVIEWS
in a complete loss of TNP1 and P1 expression, defective
chromatin condensation, and infertility (76) (Table 1).
JHDM2A is an H3K9 HDM that has a targeted action during
spermiogensis. JHDM2A binds to the core promoter regions
of TNP1 and P1 to induce their transcriptional activation by
removal of H3K9 methylation. Recently, a novel histone
modication, crotonylation, was described. It consists in the
addition of a crotonyl group (C4H5O) on the lysine residue
of all core histones (77). It marks sex chromosome, but also
gonosomes, and confers gene resistance to transcription
repressors (78).
These studies show that histone tail modications play
a crucial role in the achievement of spermatogenesis.
HISTONE-TO-PROTAMINE TRANSITION AND
MALE INFERTILITY
The transition from canonical histones to protamines is an
important step of spermiogenesis which is favored by histone
hyperacetylation (79). In humans, the P1/P2 ratio is normally
0.81.2 (80). Abnormal P1/P2 ratios and low protamine levels
are associated with increased DNA fragmentation, suggesting
that incorrect DNA compaction is more exposed to DNA dam-
age and oxidative stress (8184). Aberrant protamination
has also been linked to male infertility, because alterations
of the P1/P2 ratio are very rare in fertile men but common
in infertile men (15, 82, 85). Infertility can be associated
with either an elevated or a reduced P1/P2 ratio,
demonstrating that underexpression of both P1 and P2 are
linked to male infertility (82, 83). Low protamine levels,
resulting in abnormal elevated histone retention in sperm,
were also linked to infertility and to the fertilizing ability of
spermatozoa (86). In humans, decreased embryo quality and
poor IVF outcomes were reported with the use of sperm with
altered P1/P2 ratios (81, 83). In mouse sperm, low P2
concentrations were correlated with DNA fragmentation and
embryo death (87). This could be the consequence of
a general failure of the transition from histones to
protamines during spermiogenesis. This hypothesis is
supported by the detection of higher rates of histones and
transition protamines in sperm of patients with decreased
protamine levels or altered P1/P2 ratios. However, it is
essential to note that histone-to-protamine transition is never
complete in normal sperm and that a fraction representing
5%15% of the genome stays bound to nucleosomes
composed of canonic histones as well as histone variants
(17, 88, 89) (Fig. 1).
For a long time, the signicance of this histone retention
remained unknown and it was speculated that this was a rem-
nant of incomplete histone-to-protamine transition. How-
ever, Hammoud et al. recently demonstrated in human
spermatozoa that loci presenting histone enrichment were
not randomly distributed but were rather enriched at loci im-
portant for embryo development (13). These loci included im-
printed gene clusters, miRNA, HOX gene clusters,
developmental promoters, and signaling factors. Arpanahi
et al. also found that histone-bound DNA regions of human
sperm were associated with regulatory regions of the genome
(14). An epigenetic marking of these retained histones was
628
observed: Key developmental genes were bivalently marked
with H3K4me3 and H3K27me3, as observed in embryonic
stem cells. Additionally, H3K4me2 was enriched at develop-
mental gene promoters and H3K4me3 was enriched in HOX
regions, noncoding RNAs, and paternally imprinted loci
(13). Interestingly, histone retention was also found on genes
presenting a demethylated state, and these genes were fre-
quently coding transcription and signaling factors required
for embryo development (13).
The precise histone retention and marking and the asso-
ciated DNA demethylated state suggest that the paternal ge-
nome contains specic markers that contribute to embryo
development just after fertilization (Fig. 1). This provides an-
other epigenetic regulation in which paternal DNA is poised
for activation at specic sites necessary for embryo develop-
ment. Interestingly, a recent study by Hammoud et al. ana-
lyzed histone retention and the methylation status of DNA
in seven sperm samples: four infertile men with an abnormal
P1/P2 ratio and three men for whom the embryo development
was stopped after IVF (90). Five men had elevated histone re-
tention in their genomes, ranging from 5% to 32%. Using
ChIP experiments, two histone markers, H3K4me3 and
H3K27me3, were analyzed in this population. There was a re-
duction in the amount of H3K4me3 and H3K27 me3 at the
level of developmental transcription factor genes and some
imprinted genes. The analysis of the methylation status of im-
printed genes showed an abnormal methylation in two pa-
tients. All of these effects may have a cumulative
detrimental effect on spermatozoa fertilizing ability and on
the conceptus (90).
SPERMATOZOAL RNAs AND MALE FERTILITY
Because of its extreme compaction, the sperm nucleus has
long been thought to be inert. In fact, the spermatozoon con-
tains many specic RNAs, mRNAs, miRNAs, and piwi-
interacting RNAs (piRNAs) that could be useful to embryo
development by modifying gene expression upon fertilization
(91, 92) (Fig. 1). Reverse-transcription polymerase chain reac-
tion analysis, in situ hybridization, and oligo DNA microar-
rays have permitted identication of specic mRNAs in
mature human spermatozoa, some of them encoding prot-
amines and hormonal receptors (93). Spermatozoal RNAs
are markers of male infertility particularly concerning sper-
miogenesis. Indeed, abnormal elevated protamine mRNA
retention in the ejaculated sperm is associated with protamine
translation deregulation, protamine deciency in sperm, and
infertility (94). Levels of P1 and P2 transcripts were also re-
ported lower in the ejaculated sperm of asthenozoospermic
men (95). Moreover, a dynamic role of sperm RNAs has re-
cently been proposed. RNAs could contribute to stabilizing
the nuclear envelope and the interaction between DNA-
histones during the protamine transition. They could then
have a role in marking the DNA sequences that stay bound
to histones which is important for embryo development, as
previously described (92) (Fig. 1). Furthermore, microarray
technologies have revealed different mRNA patterns between
fertile and infertile men, which were associated with preg-
nancy rates in assisted reproduction technologies (ART)
VOL. 99 NO. 3 / MARCH 1, 2013

(9699). Interestingly, the comparison of microarray proles
between fertile and infertile men showed up- and down-
expression of several transcripts. In oligozoospermic men,
a reduced expression of genes specically involved in sper-
matogenesis and sperm function was observed (99).
CONCLUSION
Initially, epigenetic markers in male germ cells were analyzed
according to various end points. Now, DNA methylation, his-
tone modications, and sperm RNAs are found to have a close
relationship with male infertility. It seems more and more ev-
ident that various epigenetic modications associated with
infertility are all linked together. Abnormal methylation
levels are in fact associated with histone/protamine disequi-
librium as well as to RNA retention. Clearly, the sum of these
epigenetic alterations has consequences not only on male fer-
tility but also on the embryo, rst on embryo formation and
development (13) and second on the offsprings health.
Although controversial (100, 101), a rise of imprinting
diseases in children conceived by ART has been reported,
particularly Beckwith-Wiedemann and Silver Russel syn-
dromes (102). Some of the imprinted diseases observed after
ART could result from the use of male gametes with imprint-
ing defects and/or abnormal P1/P2 ratios and disturbed
histone-to-protamine ratios. However, it is not only the whole
process of ART, from hormonal stimulation to oocyte retrieval
and embryo culture that is concerned, but also infertility itself
that could affect embryo development or result in imprinting
diseases. More research is needed to evaluate the risk of trans-
mission of epigenetic abnormalities by gametes of infertile
men and women so that the risk for the conceptus can be
clearly evaluated.
Acknowledgments: The authors thank Astrid de Bantel and
Veronique Drouineaud for critical reading of the manuscript.
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