Molecular Human Reproduction, Vol. 6, No. 11, 1049-1053,
November 2000
© 2000 European Society of Human Reproduction and Embryology
Genetic diagnosis |
Study of DNA-methylation patterns at chromosome 15q11-q13 in children born after ICSI reveals no imprinting defects
1 Centre for Medical Genetics 2 Centre for Reproductive Medicine, University Hospital, Dutch-speaking Brussels Free University (Vrije Universiteit Brussel), Laarbeeklaan 101, B-1090 Brussels, Belgium
Abstract
The introduction of intracytoplasmic sperm injection (ICSI) has raised concern about safety in terms of a possible increase in the incidence of major congenital malformations, chromosomal aberrations or developmental problems. The possible influence of genetic imprinting on an ICSI procedure has not yet been investigated. We therefore studied the DNA-methylation status at a defined region in chromosome 15q11-q13 in 92 children born after an ICSI procedure. Imprinting defects in this region are associated with neurogenetic disorders, e.g. Angelman syndrome (AS) and Prader–Willi syndrome (PWS). Blood samples were taken directly after birth and stored at –80°C. Genomic DNA purification was performed from 3–7 ml EDTA–blood. Sodium bisulphite treatment was carried out in order to distinguish methylated from unmethylated DNA by transferring the unmethylated nucleic acid cytosine into uracil and leaving the methylated cytosine unchanged. Subsequently, a methylation-specific polymerase chain reaction (M-PCR) was performed. In all 92 children (83 from ICSI with ejaculated spermatozoa and nine from ICSI with non-ejaculated spermatozoa), a regular DNA-methylation pattern was found in the PWS/AS region. In none of the children were clinical symptoms of PWS or AS present. In conclusion, the results of this study do not indicate a higher risk of DNA-methylation defects in children born after ICSI.
children imprinting/DNA-methylation/ICSI/male infertility
Introduction
Intracytoplasmic sperm injection (ICSI) enables formerly infertile couples to have their own genetic child and has in many ways revolutionized the understanding of fertilization procedures. ICSI has also raised concern about its safety in terms of a possible increase in the incidence of major congenital malformations, chromosomal aberrations or developmental problems. Two groups of genetic risks can be distinguished: (i) genetic risks as a result of the possible genetic causes of infertility present in the (infertile) population: the genetic disorders causing infertility would be transmitted to the offspring and thus would be found in increased numbers in such offspring as compared with a normal fertile population; and (ii) genetic risks as a result of ICSI itself: the procedure might cause damage to the germ cells, interrupt or interfere with the natural maturation process of the sperm cells and possibly lead to genetic defects.
Most extensive studies conducted with regard to the possible genetic risks are quite reassuring. However, data from a large follow-up series reveal a slight but statistically relevant increase in sex-chromosomal and de-novo structural chromosome abnormalities (Bonduelle et al., 1996
, 1998). A possible influence of imprinting on an ICSI procedure has already been mentioned (Tesarik et al., 1998), but has not yet been investigated in a specific follow-up study. Imprinting is a mechanism of gene regulation by which only one of the parental copies of a gene is expressed (Hall, 1990; Barlow, 1995; Latham et al., 1995; Bartolomei and Tilghman, 1997; Constancia et al., 1998). Although the imprinting mechanism is largely unknown, methylation at the CpG dinucleotide is involved (Glenn et al., 1996). The germline plays a key role in the imprinting process. Imprints inherited from the previous generation have to be erased and the imprints have to be re-established according to the sex of the germ line. The exact timing of this process is elusive. In the female, methylation occurs after oocyte growth (Chaillet et al., 1991, 1995; Howlett and Reik, 1991; Ueda et al., 1992; Brandeis et al., 1994). Less is known about the male germ line. However, methylation of imprinted genes is assumed to occur between the spermatogonial and the spermatocyte stages (Bestor, 1998).
The major aim of the present study is the investigation of genetic risks of the ICSI procedure associated with DNA methylation defects. We analysed the methylation status in an imprinted region at chromosome position 15q11-q13 in children born after ICSI, as this region is known to be involved in imprinting disorders causing two distinct neurobehavioural syndromes: Prader–Willi syndrome (PWS) with a deficient or absent paternal copy and the Angelman syndrome (AS) with a deficient maternal copy at the imprinted region (Clayton-Smith and Pembrey, 1992; Wagstaff et al., 1992; Reis et al., 1994; Buiting et al., 1995; Ledbetter and Ballabio, 1995; Saitoh et al., 1997).
Materials and methods
Blood samples from 92 children born after an ICSI procedure were taken directly after birth and stored at –80°C. Genomic DNA purification was performed from 3–7 ml EDTA-blood with the QIAmp DNA Blood Maxi Kit (Qiagen, Leusden, The Netherlands).
Sodium bisulphite treatment
Before methylation-specific polymerase chain reaction (PCR), a sodium bisulphite treatment was performed, converting unmethylated cytosine to uracil and leaving methylated cytosine unchanged. In a multistep procedure, the DNA samples (2.5 µg of DNA) were first diluted in 10 mmol/l Tris pH 7.5/0.1 mmol/l EDTA in a total volume of 50 µl. Denaturation was then performed by the addition of 5.5 µl of 3 mol/l NaOH and incubation at 37°C for 15 min and 95°C for 3 min. DNA samples were put on ice. Then 500 µl of a sodium bisulphite solution (667 µl of 40 mmol/l hydroquinone, 10 ml of 2.8 mol/l sodium bisulphite and 400 µl of 10 mol/l NaOH) were added to each denaturated DNA sample, reactions were overlayed with 50 µl of Biowax (Biozym, Landgraaf, The Netherlands) and incubated for 16 h at 55°C in the dark. DNA purification was carried out using the Wizard DNA Clean-Up System (Promega, Leiden, The Netherlands). Elution was done with 50 µl of 10 mmol/l Tris pH 7.5/0.1 mmol/l EDTA at 80°C and the purified DNA was desulphonated by the addition of 5.5 µl of 3 mol/l NaOH and 15 min incubation at 37°C. Ammonium acetate (55 µl) 6 mol/l pH 7 and 220 µl of pure ethanol were added to each sample for precipitation. The washing was performed by the addition of 500 µl of 70% ethanol. After centrifugation and drying, the DNA pellet was resuspended in 25 µl of 10 mmol/l Tris pH 7.5/0.1 mmol/l EDTA.
Methylation-specific PCR (M-PCR)
PCR can take advantage of this sodium bisulphite treatment to distinguish methylated from unmethylated DNA (Kubota et al., 1997; Zeschnigk et al., 1997). The primer sequences used were as follows (Sutcliffe et al., 1994).
Maternal primer pair (primers 1 and 2 in Figure 1):
SNRPN-Mfor: 5'-TAAATAAGTACGTTTGCGCGGTC-3' (`-Mfor' standing for maternal forward)
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SNRPN-Mrev: 5'-AACCTTACCCGCTCCATCGCG-3' (`-Mrev' standing for maternal reverse)
Paternal primer pair (primers 3 and 4 in Figure 1
):
SNRPN-Pfor: 5'-GTAGGTTGGTGTGTATGTTTAGGT-3' (`-Pfor' standing for paternal forward)
SNRPN-Prev: 5'-ACATCAAACATCTCCAACAACCA-3' (`-Prev' standing for paternal reverse)
These four primers are localized in exon 1 and the 5' flanking region of the small nuclear ribonucleoprotein-associated polypeptide N (SNRPN) gene in the PWS/AS region (Zeschnigk et al., 1997). The genomic DNA represented in Figure 1 corresponds with the sequence before modification by sodium bisulphite treatment. In normal individuals, the maternal and paternal copies of the region differ in methylation of the cytosines (Cs) in the CpG dinucleotides (underlined in Figure 1): in the maternal copy Cs in the CpG dinucleotides are methylated and will remain unchanged upon bisulphite treatment, while in the paternal copy no methylation is observed and the Cs will be modified into uracil. Likewise, no methylation of Cs outside CpG dinucleotides is observed and these Cs will also be changed into uracil upon bisulphite treatment.
Each sample was analysed in two independent M-PCR reactions. PCR reactions were carried out in a 30 µl volume containing 3 µl of PCR Buffer II (Perkin Elmer), 2 mmol/l MgCl2, 2 mmol/l of each dNTP, 1 µmol/l SNRPN-Mfor and 1 µmol/l SNRPN-Mrev in the PCR reaction amplifying the maternal imprint specifically or 1 µmol/l SNRPN-Pfor and 1 µmol/l SNRPN-Prev in the paternal PCR, 0.6 IU of AmpliTaq Gold (Perkin Elmer) and 2 µl of bisulphite-modified DNA. The polymerase was activated at 95°C for 10 min. DNA was amplified in 35 cycles at 94°C, 62°C and 72°C for 30 s each, followed by a final extension at 72°C for 10 min. PCR products were separated on a 2% agarose gel, stained with ethidium bromide and visualized under UV illumination. PCR reactions without DNA or with genomic DNA from an AS patient, a PWS patient and a normal control person were also carried out at the same time. In normal individuals, amplification results in a 174 bp PCR product from the methylated maternal SNRPN allele and a 100 bp product from the unmethylated paternal allele.
Results
Clinical data
Blood samples were collected from 92 children born after an ICSI procedure. The ICSI was performed with ejaculated spermatozoa in the majority of the children (n = 83, 90.2%), with fresh testicular spermatozoa after testicular sperm extraction (TESE) in six children (6.5%) and with epididymal spermatozoa after microsurgical epididymal sperm aspiration (MESA) in three children (3.3%).
The 92 ICSI children included in the present study were born to 72 couples. Of the 92 babies, 40 were delivered from 20 twin pregnancies. The remaining 52 children were born from singleton pregnancies.
The ICSI procedure was performed because of male factor infertility in all couples. The origin of this infertility was idiopathic oligoasthenoteratozoospermia (OAT) in 61 out of 72 couples. In another three couples, obstructive azoospermia was the reason for the ICSI and non-obstructive azoospermia in another six couples. Donor spermatozoa were used in two of them. In both these cases, the father appeared to have a 47,XXY-karyotype and no spermatozoa were found in several testicular biopsies.
All 92 children participated in a clinical follow-up. None of the children (aged between 5 months and 4 years in the follow-up) had clinical symptoms of PWS, e.g. feeding problems and hypotonia in newborns, later obesity, mental retardation, short stature, craniofacial dysmorphism and hypogonadism. Symptoms indicating AS (microcephaly, severe mental retardation, jerky movements, absence of speech and paroxysms of laughter) were also absent in all children.
DNA-methylation status in the children born after ICSI
The M-PCR on genomic DNA from normal individuals showed both a 174 bp product from the maternal chromosome and a 100 bp product from the unmethylated paternal chromosome. In a PWS patient (diagnosis based on characteristic symptoms and former PCR diagnosis), the 100 bp product from the paternal chromosome was missing and in an AS patient the 174 bp product from the maternal chromosome was absent. In all 92 children both the product from the methylated maternal chromosome and that from the unmethylated paternal chromosome have been detected. A representative example of the M-PCR analysis in children born after ICSI and controls is shown in Figure 2.
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Discussion
Imprinting in the germline
The germline plays a key role in the imprinting process since it is at this stage that the existing imprints inherited from the previous generation are erased and the new imprints are established according to the sex of the germline. Little is known about the exact timing of this resetting. Global demethylation and methylation events occur in germ cells (Razin and Shemer, 1995; Huntriss et al., 1998). This also includes imprinted genes (Brandeis et al., 1994). The timing of methylation establishment in imprinted genes is clearly defined for the female but not for the male germline. In the female, oocytes are not methylated until after birth, when methylation occurs after oocyte growth (Chaillet et al., 1991; Howlett and Reik, 1991; Ueda et al., 1992; Brandeis et al., 1994). In the male, the embryonic primordial germ cells migrate into the undifferentiated gonad (Loveland and Schlatt, 1997), then differentiate into prospermatogonia and reside in a quiescent state inside the testicular seminiferous tubules. Gonadotrophic stimulation at the onset of the puberty induces spermatogenesis (the meiotic divisions giving rise to the spermatozoa) followed by spermiogenesis (the differentiation of the sperm cell, from haploid round spermatid to flagellated spermatozoa). Methylation of imprinted genes is supposed to occur during spermatogenesis between the spermatogonial and the spermatocyte stages (Bestor, 1998).
Imprinting and ICSI
Although the methylation of the imprinted genes is assumed to take place at an earlier developmental stage (spermatogenesis) than the ICSI procedure (spermiogenesis), the germ cells remain vulnerable to dysregulated de-novo methylation in the latter period (Bestor and Tycko, 1996). Especially when testicular (immature) sperm cells are injected, the ICSI might interfere with the process of imprinting or with the imprint switch and, in a worst-case scenario, provoke failures at this level.
Although the follow-up data of the ICSI children investigated in the present study do not report AS or PWS, a silent transmission to further generations cannot be completely excluded. New failures might occur in the non-expressed region and so not give rise to any immediate symptoms. If transmitted to the next generation, the subsequent offspring might have a different sex and therefore the deficient gene would be expressed. However, there is no reason to believe that only these kind of silent imprinting switch failures would occur while immediately detectable failures, as searched for in this study, would not occur.
No risk at all?
In the present study, the M-PCR of all 92 children born after an ICSI procedure revealed a methylation status in the PWS/AS region in chromosome 15q11-q13 identical to the methylation pattern in the normal controls. Signs of PWS or AS, e.g. absence of the 100 bp PCR product (PWS) from the paternal chromosome or 174 bp PCR product (AS) from the maternal chromosome, have not been found in any of the children. Therefore, the results of the present study do not indicate a higher risk of methylation defects, at least in the PWS/AS region, in the DNA of children born after an ICSI procedure. The number of investigated babies (n = 92) seems to be small when compared to the fact that in a normal population only 1 in 15 000 newborns is affected. Nevertheless, one might expect to find abnormalities in methylation patterns in at least some of these 92 children, if they did have an increased risk of DNA-methylation defects. The group of children born from ICSI with non-ejaculated spermatozoa (TESE, MESA) is of special interest, as this procedure implies fertilization with possibly immature spermatozoa. Especially in these cases, one might be concerned about whether the imprinting process is still vulnerable to dysregulation or even not yet perfectly completed at this stage of maturation. However, the small number of children from this group (n = 9) also had a regular DNA-methylation pattern in the PWS/AS region. It is also of interest to mention here that the M-PCR will not detect patients with a mosaic pattern of normal and methylation-defective cells. In these patients, paternal and maternal bands produced by the normal cells would mask the absence of unmethylated or methylated copies, respectively, in the methylation-defective cells.
In conclusion, the results of this study do not indicate a higher risk of DNA-methylation defects at the PWS/AS region in children born after an ICSI procedure. However, more data are needed, especially from children born after ICSI with non-ejaculated spermatozoa.
Acknowledgments
This work is supported by a grant to M.M. from the European Scholarship Programme (EUSP) of the European Association of Urology (EAU), and by grants from the Fund for Scientific Research Flanders (FWO-Vlaanderen). The authors would like to thank the laboratory staff at the Centre for Medical Genetics, and Mr Frank Winter of the Language Education Centre for correcting the manuscript.
Notes
3 To whom correspondence should be addressed at: Centre for Medical Genetics, University Hospital, Vrije Universiteit Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium. E-mail: lgenlsw{at}az.vub.ac.be 
References
Barlow, D.P. (1995) Gametic imprinting in mammals. Science, 270, 1610–1613.
Bartolomei, M.S. and Tilghman, S.M. (1997) Genomic imprinting in mammals. Ann. Rev. Genet., 31, 493–525.[ISI][Medline]
Bestor, T.H. and Tycko, B. (1996) Creation of genomic methylation patterns. Nature Genet., 12, 363–367.[ISI][Medline]
Bestor, T.H. (1998) Cytosine methylation and the unequal developmental potentials of the oocyte and sperm genomes. Am. J. Hum. Genet., 62, 1269–1273.[ISI][Medline]
Bonduelle, M., Wilikens, A., Buysse, A. et al. (1996) Prospective follow-up study of 877 children born after intracytoplasmic sperm injection (ICSI), with ejaculated, epididymal and testicular spermatozoa and after replacement of cryopreserved embryos after ICSI. Hum. Reprod., 11 (Suppl. 4), 131–155.
Bonduelle, M., Aytoz, A., Van Assche, E. et al. (1998) Incidence of chromosomal aberrations in children born after assisted reproduction through intracytoplasmic sperm injection. Hum. Reprod., 13, 781–782.
Brandeis, M., Frank, D., Keshet, I. et al. (1994) Sp1 elements protect a CpG island from de novo methylation. Nature, 371, 435–438.[Medline]
Buiting, K., Saitoh, S., Gross, S. et al. (1995) Inherited microdeletions in the Angelman and Prader–Willi syndromes define an imprinting center on human chromosome 15. Nature Genet., 9, 395–400.[ISI][Medline]
Chaillet, J.R., Vogt, T.F., Beier, D.R. and Leder, P. (1991) Parental-specific methylation of an imprinted transgene is established during gametogenesis and progressively changes during embryogenesis. Cell, 66, 77–83.[ISI][Medline]
Chaillet, J.R., Bader, D.S. and Leder, P. (1995) Regulation of genomic imprinting by gametic and embryonic processes. Genes Dev., 9, 1177–1187.
Clayton-Smith, J. and Pembrey, M. (1992) Angelman syndrome. J. Med. Genet., 29, 412–415.[ISI][Medline]
Constancia, M., Pickard, B., Kelsey, G.and Reik, W. (1998) Imprinting mechanisms. Genome Res., 8, 881–900.
Glenn, C.C., Saitoh, S. and Jong, M.T.C. (1996) Gene structure, DNA methylation and imprinting expression of the human SNRPN gene. Am. J. Hum. Genet., 58, 335–346.[ISI][Medline]
Hall, J.G. (1990) Genomic imprinting: review and relevance to human diseases. Am. J. Hum. Genet., 46, 857–873.[ISI][Medline]
Huntriss, J., Daniels, R., Bolton, V. and Monk, M. (1998) Imprinted expression of SNRPN in human preimplantation embryos. Am. J. Hum. Genet., 63, 1009–1014.[ISI][Medline]
Howlett, S.K. and Reik, W. (1991) Methylation levels of maternal and paternal genomes during preimplantation development. Development, 113, 119–127.[Abstract]
Kubota, T., Das, S., Christian, S.L. et al. (1997) Methylation-specific PCR simplifies imprinting analysis. Nature Genet., 16, 16–17.[ISI][Medline]
Latham, K.E., McGrath, J. and Solter, D. (1995) Mechanistic and developmental aspects of genetic imprinting in mammals. Int. Rev. Cytol., 160, 53–98.[ISI][Medline]
Ledbetter, H. and Ballabio, A. (1995) Molecular cytogenetics of contiguous gene syndromes: mechanisms and consequences of gene dosage imbalance. In Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D. (eds), The Metabolic and Molecular Bases of Inherited Disease. 7th edn, McGraw-Hill, New York, USA, pp. 811–839.
Loveland, K.L. and Schlatt, S. (1997) Stem cell factor and c-kit in the mammalian testis: lessons originating from Mother Nature's gene knockouts. J. Endocrinol., 153, 337–344.[Abstract]
Razin, A. and Shemer, R. (1995) DNA methylation in early development. Hum. Mol. Genet., 4, 1751–1755.[Abstract]
Reis, A., Dittrich, B., Greger, V. et al. (1994) Imprinting mutations suggested by abnormal DNA methylation patterns in familial Angelman and Prader–Willi syndromes. Am. J. Hum. Genet., 54, 741–747.[ISI][Medline]
Saitoh, S., Buiting, K. and Cassidy, B. (1997) Clinical spectrum and molecular diagnosis of Angelman and Prader–Willi syndrome patients with an imprinting mutation. Am. J. Med. Genet., 68, 195–206.[ISI][Medline]
Sutcliffe, J.S., Nakao, M., Christian, S. et al. (1994) Deletions of a differentially methylated CpG island at the SNRPN gene define a putative imprinting control region. Nature Genet., 8, 52–58.[ISI][Medline]
Tesarik J., Sousa M., Greco E. and Mendoza, C. (1998) Spermatids as gametes. indications and limitations. Hum. Reprod., 13 (Suppl. 3), 89–107; discussion 108–111.
Ueda, T., Yamazaki, R., Suzuki, H. et al. (1992) Parental methylation patterns of transgenic locus in adult somatic tissues are imprinted during gametogenesis. Development, 116, 831–839.[Abstract]
Wagstaff, J., Knoll, J., Glatt, K. et al. (1992) Maternal but not paternal transmission of 15q11-q13-linked nondeletion Angelman syndrome leads to phenotypic expression. Nature Genet., 1, 291–294.[ISI][Medline]
Zeschnigk, M., Lich, C., Nuiting, K. et al. (1997) A single-tube PCR test for the diagnosis of Angelman and Prader–Willi syndrome based on allelic methylation differences at the SNRPN locus. Eur. J. Hum. Genet., 5, 94–98.[ISI][Medline]
Submitted on June 2, 2000; accepted on August 21, 2000.
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