Methylation defects of imprinted genes in human testicular spermatozoa.
OBJECTIVE
To study the methylation imprinting marks of two oppositely imprinted genes, H19 and MEST/PEG1, in human testicular spermatozoa from azoospermic patients with different etiologies. Testicular spermatozoa are often used in intracytoplasmic sperm injection for treatment of male factor infertility, but the imprinting status of these cells is currently unknown.


METHODS
Experimental prospective study.


METHODS
University research laboratory and private in vitro fertilization (IVF) clinic.


METHODS
A total of 24 men, five with anejaculation, five with secondary obstructive azoospermia, five with primary obstructive azoospermia, and nine with secretory azoospermia due to hypospermatogenesis.


METHODS
Spermatozoa were isolated by micromanipulation from testicular biopsies.


METHODS
DNA methylation patterns were analyzed using bisulfite genomic sequencing with cloning analysis.


RESULTS
We found H19 complete methylation was statistically significantly reduced in secretory azoospermic patients with hypospermatogenesis, with one patient presenting complete unmethylation. Hypomethylation also affected the CTCF-binding site 6, involved in regulation of IGF2 expression. Regarding the MEST gene, all patients presented complete unmethylation although this was statistically significantly reduced in the anejaculation group.


CONCLUSIONS
Testicular spermatozoa from men with abnormal spermatogenesis carry methylation defects in the H19 imprinted gene which also affect the CTCF-binding site, further supporting an association between the occurrence of imprinting errors and disruptive spermatogenesis.
Marques
C Joana
CJ
Department of Genetics, Faculty of Medicine, University of Porto, Porto, Portugal.
Francisco
Tânia
T
Sousa
Sónia
S
Carvalho
Filipa
F
Barros
Alberto
A
Sousa
Mário
M