Human Molecular Genetics, 2007, Vol. 16, No. 21
doi:10.1093/hmg/ddm187
Advance Access published on July 17, 2007
2542–2551
Aberrant DNA methylation of imprinted loci
in sperm from oligospermic patients
Hisato Kobayashi1,2, Akiko Sato4, Eiko Otsu4, Hitoshi Hiura3, Chisako Tomatsu3,
Takafumi Utsunomiya4, Hiroyuki Sasaki5,6, Nobuo Yaegashi1,2 and Takahiro Arima3,*
1
Department of Obstetrics, 2Department of Gynecology, and 3The 21st Century COE Program, Tohoku University
Graduate School of Medicine, 2-1 Seiryo-cho, Aoba-ku, Sendai 980-8575, Japan, 4St Luke Clinic, Oita, Japan,
5
Division of Human Genetics, Department of Integrated Genetics, National Institute of Genetics, Research
Organization of Information and Systems, Mishima, Japan and 6Department of Genetics, The Graduate University
for Advanced Studies (Sokendai), Mishima, Japan
Received May 17, 2007; Revised and Accepted July 11, 2007
Recent studies suggest that assisted reproductive technologies (ART), which involve the isolation, handling
and culture of gametes and early embryos, are associated with an increased incidence of rare imprinting disorders. Major epigenetic events take place during this time and the process of ART may expose the epigenome to external influences, preventing the proper establishment and maintenance of genomic imprints.
However, the risks of ART cannot be simply evaluated because the patients who receive ART may differ
both demographically and genetically from the general population at reproductive age. In this study, we
examined the DNA methylation status of seven imprinted genes using a combined bisulphite-PCR restriction
analysis and sequencing technique on sperm DNA obtained from 97 infertile men. We found an abnormal
paternal methylation imprint in 14 patients (14.4%) and abnormal maternal imprint in 20 patients (20.6%).
The majority of these doubly defective samples were in men with moderate or severe oligospermia. These
abnormalities were specific to imprinted loci as we found that global DNA methylation was normal in
these samples. The outcome of ART with sperm shown to have an abnormal DNA methylation pattern was
generally poor. However, one sample of sperm with both paternal and maternal methylation errors used in
ICSI produced a child of normal appearance without any abnormalities in their imprinted methylation pattern.
Our data suggest that sperm from infertile patients, especially those with oligospermia, may carry a higher
risk of transmitting incorrect primary imprints to their offspring, highlighting the need for more research
into ART.
INTRODUCTION
Human-assisted reproductive technologies (ART) are important
treatments for infertile people of reproductive age, by which
the eggs and/or sperm are manipulated in the laboratory.
However, a number of studies published over the last few
years have suggested an excess occurrence of major malformation, low birth weight and other perinatal complications in
babies conceived by ART (1 –3). Furthermore, some studies
have suggested that there is an increased incidence of rare
imprinting disorders, including cases of Beckwith– Wiedemann
syndrome (BWS; NIM130650) and Angelman syndrome
(AS; NIM105830), associated with human ART (2 – 10).
Genomic imprinting, i.e. the allele-specific expression of
certain genes, accounts for the requirement for both maternal
and paternal genomes in normal development and plays
important roles in regulating embryonic growth, placental
function and neurobehavioural processes (11,12). This monoallelic expression relies on epigenetic mechanisms. DNA
methylation of CpG dinucleotides at differentially methylated
regions (DMRs) is the best-studied epigenetic mark. Imprint
resetting involves erasure of imprints in the primordial germ
cells and the acquisition of new sex specific imprints.
Although oocytes are arrested at prophase I and during the
transition from primordial to antral follicles in the post-natal
growth phase (post-pachytene), methylation is acquired
*To whom correspondence should be addressed. Tel: þ81 227177844; Fax: þ81 227177872; Email: [email protected]
# 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Human Molecular Genetics, 2007, Vol. 16, No. 21
asynchronously in a gene-specific manner in the mouse female
germ line (13,14). In the human oocyte, we previously
reported that the maternal methylation of these genes has
already been initiated to some extent in adult non-growing
human oocytes, but not in neonatal oocytes (15). In contrast,
in males, H19, Rasgrf1 and Gtl2 methylation imprints are
initiated pre-natally during embryonic germ cell development
and are completed by the pachytene phase of post-natal spermatogenesis in mice (16 –19). The imprints of gametes are
maintained stably in the early embryo, despite overall epigenetic reprogramming (20). The aberrant expression of
several imprinted genes has been linked to a number of diseases, developmental abnormalities and malignant tumours
in humans (21).
In humans, limited information is available on the methylation status of imprinted genes during gametogenesis and
embryogenesis, but the available data suggest some conservation of the timing of DNA methylation acquisition and maintenance dynamics described in mice. During normal
spermatogenesis, the erasure of methylation marks of the
maternally imprinted gene SNRPN (22) and the resetting of
the paternally imprinted gene H19 (23,24) have been reported
to be completed before germ cells enter meiosis. Marques
et al. (24) reported that there was abnormal imprinting of
only H19 in oligospermic patients and in a small number of
the normospermic patients. In contrast, appropriate genomic
imprinting was reported in spermatozoa from some infertile
men (25). However, those studies were restricted to one
specific imprinted domain.
ART involve the isolation, handling and culture of gametes
and early embryos, generally after hormone stimulation protocols. Major epigenetic events take place during this time and
the process of ART may expose the epigenome to external
influences, preventing the proper establishment and maintenance of genomic imprints (6,8). One of the issues with ART
concerns the artificial induction of ovulation with high doses
of gonadotrophins. We and others have demonstrated that
superovulation affects the methylation at certain imprinted
loci (15,26,27). The second issue is related to the culture conditions. Some studies have shown that exposure of mouse
embryos to different culture conditions can alter the expression
and imprinting of various genes, which could result in abnormal development (6 – 8). The third issue is the potential effect
of embryo cryopreservation (28,29). Experimentally, embryo
freezing has been shown to have deleterious effects on DNA,
embryonic gene expression, telomeres and plasma and
nuclear membranes. The timing of embryo transfer may also
present issues. Recent studies on monochorionic dizygotic
and conjoined twins with BWS resulting from transfer of
embryos at the blastocyst stage revealed demethylation of
LIT1 (KCNQ1OT1) (30,31), suggesting that this demethylation
occurs at a critical stage of pre-implantation development.
Furthermore, there may be other serious issues causing yet
unknown risks of ART. However, such a risk of ART treatment cannot be simply evaluated, because the patients who
receive ART may differ both demographically and genetically
from the general population at reproductive age. Usually,
patients requesting ART have a low fertility rate, an increased
reproductive loss rate and are of advanced age, all of which are
associated with various fetal and neonatal abnormalities.
2543
All these confounding factors make it difficult to evaluate
and estimate the risk of ART procedures, especially ICSI. It
is also difficult to dissect out the role of imprinting errors in
any abnormality reported after ART. To partly address these
difficulties, we determined the DNA methylation status of
the DMRs of seven imprinted genes (paternally methylated
genes: H19 and GTL2; maternally methylated genes: PEG1,
LIT1, ZAC, PEG3 and SNRPN ) directly in sperm DNA
using a combined bisulphite-PCR restriction analysis
(COBRA) and sequencing technique. The COBRA technique
gives a basic read-out for the degree of DNA methylation at
a given sequence and also acts as a control for the bisulphite
sequencing technique by confirming a lack of bias in the
cloning of the PCR product. The bisulphite technique is
more sensitive than COBRA because every potential methylation site in a target sequence is examined. We applied the
techniques to sperm DNA obtained from infertile men. Our
data showed the occurrence of methylation errors in maternal
and paternal imprinted genes that were specific to infertile
men. In addition, we found one sperm sample that contained
errors in both the paternal and the maternal methylation patterns. This sperm sample was used in an ART procedure
that led to a successful and normal pregnancy and birth.
This gave an opportunity to examine the DNA methylation
status of the seven imprinted genes in the baby.
RESULTS
Analysis of the paternal methylation imprint
on the sperm of infertile couples
We analysed the primary DNA methylation pattern of seven
imprinted loci in 97 sperm DNA samples from male patients
who were within couples reporting fertility problems. We
examined a total of 18 CpG sites in a 220 bp fragment of
H19 and 15 CpG sites in a 259 bp fragment of GTL2
(Fig. 1Aa and b). These two genes are normally expressed
only from the maternal allele and are linked to paternally
methylated DMRs.
To confirm that the sequencing results from the limited
number of templates accurately reflected the overall methylation pattern of the amplified sequences from the isolated
germ cell populations, we carried out restriction analysis
(COBRA). Germ cell and somatic cell genomic DNA was cut
with enzymes that could cleave only the methylated templates
of the same bisulphite-treated PCR samples that were used for
cloning and sequencing. PCR of these DMRs was followed by
digestion with the enzymes TaqI and MluI for H19 and with
TaqI and NruI for GTL2, so that the undigested and digested
products indicated unmethylated and methylated templates,
respectively. About half methylated and half unmethylated
templates, representing paternal and maternal alleles, were
obtained after the treatment of DNA from normal somatic leukocytes, indicating a lack of bias in the PCR (Fig. 1Ba and b,
control). On all DNA from sperm that had a normal count
and were motile, H19 was shown to yield the digested methylated band using the method of COBRA, suggesting that the
majority of the sample was methylated at this locus.
However, in four cases of oligospermia (moderate oligospermia, one case and severe oligospermia, three cases),
2544
Human Molecular Genetics, 2007, Vol. 16, No. 21
Figure 1. Methylation status of imprinted genes in genomic DNA prepared from ejaculated human sperm. (A) Genomic structures of the human DMRs of
H19(a), GTL2(b), PEG1(c), LIT1(d), ZAC(e), PEG3(f) and SNRPN(g) and the repetitive sequence non-imprinted genes LINE1(h) and Alu(i). The extent of
the regions analysed in this study and the GenBank accession numbers are shown under the line. Filled boxes and horizontal arrows indicate the genes and
orientation, respectively. Open boxes represent the DMRs of the genes. Arrowheads above the CpGs indicate which of these sites are contained within a
repeat. The horizontal arrows represent the primers. Vertical arrows indicate the unique bisulphite-PCR restriction enzyme sites analysed in (B) T, TaqI; Ml,
MluI; N, NruI; Mb, MboI and H, HhaI. The vertical bars represent a CpG site. (B) Methylation imprint errors in the sperm from ART male patients.
Overall methylation status of the DMRs (by COBRA) in the adult sperm DNA and in the control leukocyte DNA. The same bisulphite-treated DNA amplified
by PCR and used for (A) was digested with restriction enzymes that cut only if the site was methylated at the positions indicated in (A). Sizes of digested fragments are indicated on the right. For H19 (a), case 26 showed the unmethylated band and for PEG1 (b), cases 62, 63 and 66 showed the methylated band.
(C) Bisulphite-PCR sequencing for PEG1 cases 62, 63, 64 and 66. Closed and open circles represent methylated and unmethylated CpGs, respectively. The
results are summarized in Table 1.
unmethylated bands were found. Their sequences were then
determined and gave similar results to the COBRA (Figs 1Ba
and 2Ba, sperm; Table 1). This result was similar to that of
a previous report (24). We next performed the methylation
analyses of GTL2. The COBRA assay for GTL2 showed it to
be methylated in almost all male germ lines. However, as
was found for H19, six sperm samples with oligospermia (moderate oligospermia, two cases and severe oligospermia, four
Human Molecular Genetics, 2007, Vol. 16, No. 21
2545
Figure 2. DNA methylation analyses in a case of a neonate whose father’s sperm showed abnormal methylation at imprinted loci. DNA methylation analyses
COBRA (A) and bisulphite-PCR sequencing (B) of genomic DNA prepared from the neonate produced by ICSI treatment with sperm (case 26) that we showed
had an abnormal pattern of DNA methylation at imprinted loci. Sp, sperm DNA from the male patient; Bl, leukocyte DNA from the male patient and Um, leukocyte DNA from the umbilical cord blood from the neonate. Closed and open circles represent methylated and unmethylated CpGs, respectively.
cases) showed the unmethylated bands (Table 1, data not
shown). We also carried out the bisulphite-PCR sequencing
of the CpG sites of H19 and GTL2. When we examined the
data for both H19 and GTL2, we found one case that showed
abnormal methylation of both the H19 and GTL2 DMRs. This
individual had severe oligospermia. Surprisingly, there were
five cases that appeared normal at the level of microscopic
examination but showed an abnormal unmethylated pattern.
Nonetheless, the occurrence of abnormal methylation at the
H19 and GTL2 loci was significantly increased in oligospermic
patients when compared with normospermic patients (Supplementary Material, Table S1).
Analysis of the maternal methylation imprint of the
sperm
We next extended the methylation analyses of the human sperm
DNA to maternal methylation regions associated with PEG1,
LIT1, ZAC, PEG3 and SNRPN. We previously reported that
these regions of PEG1, LIT1, ZAC were DMRs that showed
50% methylation in somatic cells and were completely methylated in the fully grown ovum (15). Here, we confirmed that the
human SNRPN and PEG3 DMRs in both normal somatic and
germ cells were methylated in a similar manner as previously
shown (data not shown). We then performed the methylation
analyses of the five maternal DMRs in the sperm samples.
First, COBRA analyses were performed in order to examine
the overall methylation patterns (Fig. 1Bb). Almost all sperm
samples were shown to have the expected unmethylated
pattern, but a few samples also had the methylated band
pattern (PEG1 12 cases; LIT1 four cases; ZAC three cases;
PEG3 five cases and SNRPN four cases) (Table 1). Furthermore, as with the paternal DNA methylation analysis, the proportion of oligospermic patients with aberrant methylation
profiles was significantly increased when compared with normospermic patients (Supplementary Material, Table S1).
We confirmed the results of the COBRA assay by sequencing
10 –20 clones at every CpG site within the DMRs (Fig. 1C and
Table 1). Half of the sperm samples with severe oligospermia
(five of 10 cases) were shown to have an abnormal methylation
pattern in the maternal DMRs. Among the cases of moderate
oligospermia, three of eight cases were shown to have an abnormal maternal methylation pattern. Also, 12 cases in which the
sperm appeared normal were shown to have both methylated
and unmethylated band patterns.
Among the cases examined, 12 of 24 had an abnormal
methylation pattern. Ten cases were found to show aberrant
methylation at both paternally and maternally methylated
DMRs. Of the six severest oligospermia cases, five were
shown to have both the maternal and the paternal abnormal
methylation patterns. Within the normal-appearing sperm
samples, very few showed aberrant methylation at both paternally and maternally methylated DMRs.
Methylation analysis of non-imprinted repetitive genes
The great majority of CpGs present in the mammalian genome
are contained within repetitive DNA elements. To find whether
2546
Human Molecular Genetics, 2007, Vol. 16, No. 21
Figure 2. Continued.
DNA methylation errors occur in ejaculated sperm on a more
global level, we assessed the methylation profile of nonimprinted repetitive elements, such as long interspersed
nucleotide elements (LINE1) and Alu elements. We examined
a total of 28 CpG sites in a 413 bp fragment of LINE1 and
12 CpG sites in a 152 bp fragment of Alu in sperm that we
had identified with abnormal methylation at imprinted loci
by COBRA (Fig. 1Ah and i). Our results showed that the
ratio of the methylation showed no significant differences
between the cases of normal imprint methylation and those
of aberrant imprint methylation (Table 1). All the results of
the COBRA and bisulphite-PCR analyses are shown in Supplementary Material, Table S2.
Analysis of an infant born from ICSI from a patient
with an aberrant sperm DNA methylation pattern
We followed the infertility treatments and pregnancy history
of 24 cases that showed the abnormal DNA methylation patterns (Table 2). Four of the pregnancies from ICSI ended in
a miscarriage. One patient had spontaneous pregnancy
without ART. Two patients after ICSI had live births. In one
of these cases, the baby was delivered at full term by a caesarean operation at the patient’s request. The neonate was female
and birth weight was 2650 g, height 47.2 cm and Apgar score
9 points/1 min (10 points/5 min). No abnormalities were seen
on physical and neurological examinations.
We were able to examine whether the aberrant imprint
methylation of H19, PEG1 and ZAC that we had identified
in the paternal germ cells was transmitted to the infant by carrying out the bisulphite-PCR methylation assays of the seven
imprinted loci in DNA obtained from umbilical cord blood
from the infant and also in the father’s blood (Fig. 2). In the
amplified region of H19 DMR (AF125183: 7877 – 8096),
both samples were heterozygous for a C/A polymorphism at
nucleotide 8008, which allowed us to differentiate between
the two parental alleles by bisulphite-PCR sequence. This
showed that there was allele-specific DNA methylation and
we infer that the paternal H19 allele was methylated. There
was no polymorphism in the other amplified regions;
Human Molecular Genetics, 2007, Vol. 16, No. 21
2547
Table 1. COBRA and bisulphite-PCR analyses of methylation profiles of seven imprinted and two non-imprinted genes in infertile sperm sample sequences
Case (age)
Microscopic examination
H19
GTL2
PEG1
LIT1
ZAC
PEG3
SNRPN
LINE-1
Alu
30 (36)
32 (33)
37 (34)
39 (31)
49 (38)
52 (31)
54 (32)
56 (34)
59 (33)
66 (34)
79 (30)
85 (34)
87 (32)
90 (38)
7 (46)
29 (34)
31 (40)
62 (40)
16 (34)
26 (38)
63 (27)
67 (37)
82 (37)
83 (32)
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Moderate
Moderate
Moderate
Moderate
Severe
Severe
Severe
Severe
Severe
Severe
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
23.7 (11.4)
þ
þ
þ
þ
10.7 (10.6)
59.0 (55.6)
89.4 (80.7)
þ
þ
43.5 (51.2)
þ
91.8
þ
88.9
þ
88.4
89.5
þ
þ
þ
þ
þ
þ
þ
þ
75.4
88.1
52.1 (75.8)
þ
89.2
þ
82.0
78.1
17.9 (12.7)
19.8 (21.9)
17.2 (18.4)
2
2
2
8.0
2
10.6
18.8 (42.1)
2
17.0 (21.2)
2
2
33.4 (43.4)
2
2
9.6 (8.7)
2
52.3 (60.5)
17.8 (20.8)
16.7 (37.3)
2
2
2
2
2
2
2
10.5 (3.0)
2
2
10.2 (1.8)
8.1
2
2
2
2
2
2
2
2
2
2
13.2 (19.0)
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
21.9 (19.5)
2
2
12.9 (13.7)
16.6 (14.3)
2
2
2
12.3
2
2
13.1 (4.5)
2
2
2
11.9
2
12.1
2
2
2
2
12.8
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
8.3
13.3 (5.0)
8.9
2
2
2
2
2
2
2
8.4
44.9
43.7
56.0
47.3
46.7
45.3
46.7
55.9
50.3
46.9
46.8
44.1
46.6
53.7
48.0
49.1
54.1
44.3
56.1
52.8
53.0
52.4
50.7
46.9
22.7
22.7
20.2
20.1
24.3
22.8
22.2
24.0
20.5
22.1
19.4
22.9
19.7
20.8
19.8
21.9
20.9
21.7
22.6
21.3
20.0
19.6
18.9
21.0
Methylation results: þ, almost fully methylated; 2, almost fully unmethylated. Number represents percent methylated with bisulphite result given in
brackets. Moderate: moderate oligospermia (sperm count 5 – 20 106/ml), severe: severe oligospermia (,5 106/ml), normal: normal spermia
(20 106/ml). The results of all cases were shown in detail in Supplementary Material, Table S2.
Table 2. Treatment and outcome of ART with sperm shown to have abnormal DNA methylation patterns at imprinted gene loci
Case (age)
Cause of infertility
ART (number)
Fertilized rate (%)
Embryo stage
Embryo grade
Pregnancy (time)
Abortion (time)
7 (46)
16 (34)
26 (38)
29 (34)
30 (36)
31 (40)
32 (33)
37 (34)
39 (31)
49 (38)
52 (31)
54 (32)
56 (34)
59 (33)
62 (40)
63 (27)
66 (34)
67 (37)
79 (30)
82 (37)
83 (32)
85 (34)
87 (32)
90 (38)
Unknown
Male factor
Male factor
Tubal factor
Unknown
Unknown
Endometriosis
Tubal factor
Endometriosis
Unknown
Tubal factor
Male factor
Unknown
Unknown
Unknown
Male factor
Male factor
Endometriosis
Unknown
Endometriosis
Male factor
Unknown
Unknown
Unknown
IVF (1), ICSI (10)
ICSI (2)
ICSI (21)
IVF(1), ICSI (4)
ICSI (2)
ICSI (1)
IVF (7), ICSI (3)
ICSI (3)
IVF (1), ICSI (3)
IVF (2), ICSI (2)
ICSI (1)
ICSI (1)
0
IVF (1), ICSI (1)
ICSI (1)
ICSI (2)
0
IVF (2)
0
IVF (2)
0
0
IVF (1), ICSI (1)
0
50.6
83.5
68.3
77.8
66.3
0
67.3
80
65.7
49.8
50
40
—
81.5
69
45
—
33
—
33
—
—
82
—
6 cell
3
8 cell
9 cell
6 cell
2
2
2
—
1
1a
—
—
1
—
—
5 cell
3
8 cell
2
—
—
—
1
1b
—
1
1
—
—
1a
—
—
—
—
—
—
—
—
—
—
—
—
1
—
—
—
—
—
—
—
—
—
—
1
—
a
ICSI pregnancy and birth.
Spontaneous pregnancy and birth.
b
however, we observed about half methylated and half
unmethylated (or partially demethylated in GTL2) templates
likely to represent the differentially methylated paternal and
maternal alleles both by COBRA and by bisulphite-PCR
sequencing. Normal methylation patterns were seen at all
the seven DMRs of the imprinted genes and the repetitive
2548
Human Molecular Genetics, 2007, Vol. 16, No. 21
elements that we examined in the infant’s DNA. These results
showed that the abnormal methylation pattern seen in the
father’s sperm DNA was not inherited by the neonate in this case.
DISCUSSION
Imprint region-specific methylation error
in the sperm in infertile patients
The DNA methylation of DMRs associated with imprinted
genes is reset with every reproductive cycle. Imprint resetting
involves the acquisition of new sex-specific imprints. In
humans, ejaculated and mature sperm should be methylated
in the paternal DMRs, but unmethylated in the maternal
DMRs. We analysed the DNA methylation status of seven
DMRs in the ejaculated sperm of men from 97 couples who
had undergone infertility treatment. We found 14 samples
with abnormal methylation of the paternal DNA methylation
at H19 and GTL2 and 20 samples with abnormalities of
maternal DMRs at PEG1, LIT1, ZAC, PEG3 and SNRPN. Of
these, approximately half of the cases showed abnormalities
in both maternal and paternal imprints. The majority of
these doubly defective samples were in men with moderate
or severe oligospermia. These abnormalities were specific to
imprinted loci because we found that global DNA methylation
was normal in these samples.
A limited number of studies have been reported on the
profile of DNA methylation acquisition of DMRs of the
imprinted genes in human adult sperm. Marques et al. reported
on the association of the abnormal genomic imprinting of H19
with hypospermatogenesis. The methylation aberration in the
CTCF binding site (23) of the H19 gene was seen in the
cases of moderate oligospermia (12.5%) and was seen even
more frequently in cases of severe oligospermia (30%), but
was not seen in normospermia patients (24). Our results for
H19 were similar to those findings reported. However, the
imprint methylation errors were not limited to H19 and were
also seen in the normal-appearing sperm.
In mice, H19, Rasgrf1 and Gtl2 methylation imprints are
initiated pre-natally at almost the same time during embryonic
germ cell development. We identified the equivalent human
DMR of GTL2, but could not find the human RASGRF1
DMR within in the CpG-rich region analysed. In our sperm
analysis, we found incomplete methylation of GTL2; normospermia (6.3%) (five of 79 cases), moderate oligospermia
(25%) (two of eight cases) and severe oligospermia (50%)
(five of 10 cases). These results reveal that abnormal spermatogenesis (leading to low sperm counts) is associated with
defective GTL2 methylation. One patient with severe oligospermia, among the 97 patients examined, showed incomplete
methylation acquisition in both H19 and GTL2 DMRs. As
paternal methylation was absent, the abnormal genomic
imprinting could be a result of a change in the DNA methyltransferase activity (32), and the differences between H19
and GTL2 DMRs may indicate the specificity and sensitivity
of each imprinted region.
When we examined the methylation acquisition of the
maternally methylated DMRs in the ejaculated sperm, we
found that errors were more frequent than those at paternally
methylated DMRs. A total of 20 of 97 cases (20.6%) were
shown to have abnormal maternal methylation of PEG1,
LIT1, ZAC, PEG3 and SNRPN. Our results showed the presence of maternal methylation in cases of normospermia
(15.2%) (12 of 79 cases), moderate oligospermia (37.5%)
(three of eight cases) and severe oligospermia (50%) (five of
10 cases). The results suggest that abnormal spermatogenesis
was associated with a rise in the maternal methylation or a
failure in erasure. The presence of abnormal DNA methylation
at different maternal DMRs varied between samples in many
cases. This may suggest differences in the specificity and sensitivity of each imprinted region. As the global methylation
was stable, the DMRs of imprinted genes are more labile
and readily changeable. It has been shown that the short
tandem repeat elements within the DMRs of some imprinted
genes are important for imprinting (33). We speculate that
the degree to which imprinted genes are repeat-like may be
one of the factors involved in determining methylation
status. However, we cannot test this hypothesis.
The most frequent methylation error was seen in the PEG1
DMR. In our previous report, we showed that demethylation
of PEG1 was present in the growing oocytes from superovulated infertile women (15). This PEG1 DMR may be
especially vulnerable to errors. In both humans and mice,
the PEG1 DMR spans the promoter, the first exon and part
of the first intron and is unmethylated on the active paternal
allele (34,35). Paternal transmission of a methylated Peg1
gene results in growth-retarded embryos and increased postnatal death. Abnormal adult maternal behaviour has also
been noted in Peg1-deficient females (36). A number of
imprinted genes have also been shown to play a role in regulating neonatal growth and development (MRC Mammalian
Genetics Unit, Harwell, UK, http://www.mgu.har.mrc.ac.uk/
research/imprinting/function.html). In general, ART-treatment
babies are characterized by low birth weight. Our work provides further data to suggest that this may be a result of accumulated small changes in DNA methylation at imprinted loci
in the sperm and oocytes of infertile couples.
Mechanisms of methylation errors in sperm
Our results showed partial methylation errors in almost all
cases. This acquisition of errors had no specificity and
showed a scattered distribution in the various imprinted loci.
DNA methyltransferases (Dnmts) and methyl-binding
domain proteins are probably key regulators in the process
of methylation acquisition of the germ cells (37 – 42). The
Dnmt 3A and 3L knockout female mice were aborted and
could not acquire the maternal imprint methylation. In
males, the phenotype was oligospermia. In humans with infertility problems, there are no reports as yet that these de novo
methylases or associated proteins are lost or mutated.
A model based on errors in trafficking of the oocyte isoform
of DNMT1 has been proposed to explain the genetics of
BWS in monozygotic twins (43). Recently, Arnaud et al.
(44) showed that a maternal imprint could be acquired in the
absence of Dnmt3L in female germ cells. This incomplete
penetrance of DNMT3L deficiency was neither locus nor
embryo-specific, but instead stochastic, suggesting that in
the absence of Dmnt3L, other factors can mark individual
DMRs. In addition, natural changes in gene expression
Human Molecular Genetics, 2007, Vol. 16, No. 21
levels occur during ageing, such as changes in the expression
of Dnmt3 proteins required for the establishment of the germ
line imprint (41), which may, in turn, be due to changes in the
endocrine environment.
Outcome of using sperm with abnormal imprint
methylation
We found 24 patients whose sperm had abnormal DNA
methylation among the sperm samples of men from 97 infertile couples. Although the sample size was relatively limited
(24), the live-birth rates achieved by ICSI technology within
this group appeared lower than expected from this technology.
Only one sperm sample with both paternal and maternal
methylation errors of imprinted genes was successfully used
to fertilize an ovum using ICSI and resulted in normal pregnancy. The newborn was normal and did not show any
abnormality in the methylation of imprinted genes. There
are two possible explanations: one is that the fertilizing
sperm prepared using ICSI was a rare sperm with a normal
DNA methylation pattern and the other is that the methylation
abnormalities reverted to normal after fertilization. We cannot
distinguish between these two possibilities. However, we are
now analysing the methylation status of imprinted genes in
the DNA of the abortion products resulting from fertilization
with other sperm sample shown to have with abnormal DNA
methylation patterns. This may allow us to partially resolve
this question.
In addition to general growth abnormalities, many imprint
methylation errors also lead to the occurrence of various
cancers (45,46). Although our study examined a limited
number of imprinted genes, our data, and those of others,
strongly support the need for further research in this area.
A retrospective examination of children born after each ART
method focusing on imprinted genes would also be valuable
in determining the safest and most ethical approach to use.
MATERIALS AND METHODS
Sperm collection
The ejaculated sperm samples were collected from the male
partner of 97 infertile couples who had consulted a physician
at St Luke Clinic. Routine semen analysis (volume, counting,
rates of motility and occupied acrozorm) was performed.
Motile sperm cells were purified away from lymphocyte contamination, immature germ cells and epithelial cells in using
the swim-up method (47). Of the 97 patients, 79 showed a
normal sperm count (.20 106/ml). The remaining
18 patients had oligospermia (10 cases had severe oligospermia).
The study was performed after obtaining patients’ consent and
with approval of the institutional Ethics Committee. The
sperm were washed repeatedly and placed in phosphatebuffered saline and DNA was obtained by using a standard
extraction method with the addition of 0.1 mM 2mercaptoethanol (48). Normal human leukocyte DNA was
used as a control.
2549
Bisulphite treatment PCR
The methylation assay was performed at the DMRs of seven
imprinted genes [H19, GTL2, PEG1 (MEST ), LIT1
(KCNQ1OT1), ZAC (PLAGL1), PEG3 and SNRPN ] in
humans using the COBRA and sequencing technique (19).
Each sperm DNA sample was treated with sodium bisulphite
using an EZ DNA Methylation Kit (Zymo Research,
Orange, CA) and amplified by PCR as follows: a PCR reaction
mix containing 0.5 mM of each of the four following primer
sets, 200 mM dNTPs, 1 PCR buffer, 1.25 U of EX Taq Hot
Start DNA Polymerase (Takara Bio, Tokyo, Japan) in a total
volume of 20 ml was used. The following PCR programme
was used for PEG1 and LIT1: 1 min of denaturation at 948C
followed by 35 cycles of 30 s at 948C, 30 s at 608C and 30 s
at 728C and a final extension for 5 min at 728C. In the case
of H19 and SNRPN, semi-nested PCR was carried out as
DNA input for 1 ml of the first-round PCR product with the
same first-round PCR condition. The PCR conditions for
GTL2, ZAC and PEG3 were described previously (49 – 51).
The region analysed for each of these genes was within the
DMR of CpG islands. We examined 18 CpG sites in a
220 bp fragment of H19 (AF125183: 7877 –8096), 15 CpG
sites in a 259 bp fragment of GTL2 (AL117190: 51004 –
51262), 22 CpG sites in a 219 bp fragment of PEG1
(Y10620: 609 –827), 26 CpG sites in a 307 bp fragment of
LIT1 (U90095: 67252 – 67558), 19 CpG sites in a 152 bp fragment of ZAC (AL109755: 52735 –52886), 33 CpG sites in a
322 bp fragment of PEG3 (AC006115: 163172 – 163493) and
21 CpG sites in a 240 bp fragment of SNRPN (U41384:
15256 – 15495). These are summarized in Table 3.
To confirm that the sequencing results did not reflect a
cloning bias, restriction analysis (COBRA) was carried out
on germ cell and somatic cell DNA, cutting the DNA with
enzymes that could cleave only the methylated templates of
the same bisulphite-treated PCR samples. PCR products of
each DMR were then digested with the enzyme TaqI for
H19, GTL2, PEG1, LIT1, ZAC, PEG3 and SNRPN, with
MluI for H19, with NruI for GTL2 and with MboI for
PEG1, so that the undigested and digested products indicated
unmethylated and methylated templates, respectively, and
electrophoresed on a 2.5% agarose gel. If the unmethylated
bands were seen in H19 and GTL2 paternally imprinted
genes or the methylated bands were seen in PEG1, LIT1,
ZAC, PEG3 and SNRPN maternally imprinted genes, each gene
was quantified with Lumiimager analyser and Lumianalyst
software package (Roche Diagnostics, Basel, Switzerland),
and the percentage of methylated restriction enzyme site in
each genomic sample was calculated from the ratio between
the enzyme-cleaved PCR products and the total amount of
PCR products. The PCR products were purified and cloned
into the pGEM-T vector (Promega, Madison, WI, USA), and
individual clones were sequenced using M13 reverse primer
and an automated ABI Prism 3130xl Genetic Analyser
(Applied Biosystems, Foster city, CA, USA). An average of
20 clones was sequenced for each individual. The repetitive
sequences (LINE1 and Alu) were also examined using the
same methods. The PCR conditions were previously described
(52) and the restriction enzymes used were HinfI and MboI,
respectively. At least two separate sodium modification
2550
Human Molecular Genetics, 2007, Vol. 16, No. 21
Table 3. Sequences of primers using the bisulphite-PCR analyses
Gene
Paternally methylated genes
H19a
GTL2
Maternally methylated genes
PEG1
LIT1
ZAC
PEG3
SNRPNa
Non-imprinted genes
LINE-1
Alu
Primer sequence (50 –30 )
Size (bp)
Number of CpG sites
F1:
F2:
R1:
F:
R:
AGGTGTTTTAGTTTTATGGATGATGG
TATATGGGTATTTTTGGAGGTTTTT
ATAAATATCCTATTCCCAAATAACCCC
GGGTTGGGTTTTGTTAGTTGTT
CCAATTACAATACCACAAAATTAC
220
18
259
15
F:
R:
F:
R:
F:
R:
F:
R:
F1:
F2:
R1:
TYGTTGTTGGTTAGTTTTGTAYGGTT
CCCAAAAACAACCCCAACTC
TTTTGGTAGGATTTTGTTGAGGAGT
CCTCACACCCAACCAATACCTC
GGGGTAGTYGTGTTTATAGTTTAGTA
CRAACACCCAAACACCTACCCTA
AAAAGGTATTAATTATTTATAGTTTGGT
AAAAATATCCACCCTAAACTAATAA
GTGTTGTGGGGTTTTAGGGGTTTAG
AGGGAGTTGGGATTTTTGTATTG
CTCCCCAAACTATCTCTTAAAAAAAACC
219
22
307
26
152
19
Kamikihara et al. (50)
322
33
El-Maarri et al. (51)
240
21
F:
R:
F:
R:
TTGAGTTGTGGTGGGTTTTATTTAG
TCATCTCACTAAAAAATACCAAACA
GATCTTTTTATTAAAAATATAAAAATTAGT
GATCCCAAACTAAAATACAATAA
413
28
Yang et al. (52)
152
12
Yang et al. (52)
References
Kawakami et al. (49)
a
Hemi-nested PCRs were carried out in these regions. F1 and R1 were used for first PCR and F2 and R1 were used for second PCR.
treatments were carried out for each DNA sample and at least
three independent amplification experiments were performed
for each individual examined.
Statistical analysis
Two proportions were used to analyse the observed data using
the difference between two proportions test (STATISTICA,
StatSoft, Tokyo, Japan). P-values less than 0.05 were considered significant.
SUPPLEMENTARY MATERIAL
Supplementary Material is available at HMG Online.
ACKNOWLEDGEMENTS
We would like to thank Miss M. Nasu for technical assistance
and all the members of our laboratory for their support and
valuable suggestions. In particular, we thank Dr R. John for
comments on the manuscript.
Conflict of Interest statement. No conflicts of interest were
reported by the investigators.
FUNDING
This work was supported by a grant from the Ministry of
Health and Welfare of Japan (19390423 and 19791131),
Suzuken Memorial Foundation, Akaeda Medical Research
Foundation and Smoking Research Foundation (T.A.),
a grant-in-aid from Kurokawa Cancer Research Foundation,
Scientific Research on Priority Areas from the Ministry of
Education, Science and Culture, Japan, the Ministry of
Health, Labour and Welfare, Japan and 21st Century COE
Program Special Research Grant (Tohoku University) from
the Ministry of Education Science, Sports and Culture,
Japan (N.Y.).
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