Abstract (EN):
In the present study, we evaluated the methylation status of H19 gene DMR (Differentially Methylated Region) using sodium bisulphite treatment of DNA, cloning of PCR (Polymerase Chain Reaction) products and automated DNA sequencing. We studied 69 clones from 5 sperm samples obtained from males with normal sperm counts (¿ 20 x 106 Sz/mL) and 31 clones from 2 sperm samples isolated from patients with severe oligozoospermia (< 5 x 106 Sz/mL). In sperm cells from severe oligozoospermic patients, we observed that the majority of clones (26/31, 83.9%) presented incomplete methylation, in contrast to clones obtained from sperm from normozoospermic males (33/69, 47.8%). In severe oligozoospermia, the average of unmethylated CpGs was of 4.1, ranging between 1 to 17 affected CpGs, while in normozoospermia the average was of 0.7, ranging from 1 to 3 affected CpGs, being statistically significant the difference between the two groups (p=0.001). The simultaneous demethylation of multiple CpGs occurred in 5/33 (15.2%) of the clones with incomplete methylation in the normozoospermia group, while in severe oligozoospermia the majority of the cases (19/26, 73.1%) presented associated demethylation simultaneously. Concomitantly, for each CpG position, the level of methylation deficit in normozoospermia was always inferior than 10% with the exception of CpG 6 and 9 (12 and 14%, respectively), while in severe oligozoospermia the majority of cases presented values of demethylation superior than 20% (P = 0.000-0.044). The complete demethylation of CTCF protein binding site (CpG 4-8) was only found in severe oligozoospermic cases (3/31, 9.7%). In conclusion, sperm from severe oligozoospermic patients present high rates of genomic imprinting errors, leading to the possibility of transmission of these errors to the offspring during ICSI (Intracytoplasmic Sperm Injection) treatments. In the absence of methylation at H19 DMR, CTCF protein might bind to the paternal allele and, consequently, promote the transmission of two active H19 and two inactive IGF2 alleles. This situation could negatively affect the in vitro development of preimplantation embryo. Thus, these results suggest the importance of a previous study of genomic imprinting in sperm before the ICSI procedure, in order to evaluate the existence of any risk. © 2005 Sociedad Española de Andrología.
Idioma:
Espanhol
Tipo (Avaliação Docente):
Científica