During spermiogenesis, histone-to-protamine exchange causes chromatin condensation. Spermatozoa from infertile men are known to exhibit an increased protamine-1 (PRM1) to protamine-2 (PRM2) protein ratio. Since patients undergoing testicular sperm extraction (TESE) followed by intracytoplasmic sperm injection (ICSI) reveal low fertilization rates, whether the outcome of ICSI could be related to the percentage of round spermatids expressing PRM1-mRNA and PRM2-mRNA was investigated. Applying in-situ hybridization, 55 testicular biopsies from men undergoing TESE/ICSI were investigated. The percentage of PRM1-mRNA and PRM2-mRNA positive spermatids was significantly (P < 0.0001) decreased in men with at least qualitatively normal spermatogenesis (PRM1-mRNA: 58.4 +/- 13.8%; PRM2-mRNA: 56.4 +/- 11.3%) and impaired spermatogenesis (PRM1-mRNA: 32.6 +/- 10.8%; PRM2-mRNA: 31.7 +/- 11.1%) compared with men with obstructive azoospermia and quantitatively normal spermatogenesis (PRM1-mRNA: 79.9 +/- 4.6%; PRM2-mRNA: 78.1 +/- 5.7%). A positive correlation (r(PRM1) = 0.733; r(PRM2) = 0.784; P < 0.001) was demonstrated between the score and the percentage of PRM1-mRNA and PRM2-mRNA positive spermatids. While successful fertilization was neither related to the score, nor to the percentage of PRM1-mRNA and PRM2-mRNA positive spermatids, a significant (P < 0.05) relationship was demonstrated between successful fertilization and the PRM1-mRNA to PRM2-mRNA ratio. Therefore, the PRM1-mRNA to PRM2-mRNA ratio in round spermatids may serve as a possible predictive factor for the outcome of ICSI.
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 EDTAblood. 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 DNAmethylation defects in children born after ICSI. Key words: children imprinting/DNA-methylation/ICSI/male infertility Introduction parental copies of a gene is expressed (Hall, 1990; Barlow, 1995; Latham et al., 1995; Bartolomei and Tilghman, 1997; Intracytoplasmic sperm injection (ICSI) enables formerly infer- Constancia et al., 1998). Although the imprinting mechanism tile couples to have their own genetic child and has in is largely unknown, methylation at the CpG dinucleotide is many ways revolutionized the understanding of fertilization involved (Glenn et al., 1996). The germline plays a key role procedures. ICSI has also raised concern about its safety in in the imprinting process. Imprints inherited from the previous terms of a possible increase in the incidence of major congenital generation have to be erased and the imprints have to be remalformations, chromosomal aberrations or developmental established according to the sex of the germ line. The exact problems. Two groups of genetic risks can be distinguished:timing of this process is elusive. In the female, methylation (i) genetic risks as a result of the possible genetic causes of occurs after oocyte growth (Chaillet et al., 1991(Chaillet et al., , 1995; Howlett infertility present in the (infertile) population: the genetic and Reik, 1991;Ueda et al., 1992; Brandeis et al., 1994). Less disorders causing infertility would be transmitted to the offis known about the male germ line. However, methylation of spring and thus would be found in increased numbers in such imprinted genes is assumed to occur between the spermatogonoffspring as compared with a normal fertile population; and ial and the spermatocyte stages (Bestor,...
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