DNA methylation imprinting errors in spermatogenic cells from maturation arrest azoospermic patients

Abstract

Imprinting errors have been described in spermatozoa from infertile patients with oligozoospermia and azoospermia. However, little is known about methylation of imprinted genes in other spermatogenic cells from azoospermic patients. Therefore, we aimed to evaluate the methylation status of single CpGs located in the differentially methylated regions (DMRs) of two imprinted genes, one paternally (H19) and one maternally (MEST) methylated, in primary spermatocytes of azoospermic patients presenting complete (MAc, n = 7) and incomplete (MAi, n = 8) maturation arrest, as well as in other spermatogenic cells from MAi patients that presented focus of complete spermatogenesis in some seminiferous tubules. We observed H19 imprinting errors in primary spermatocytes from one MAi patient and MEST imprinting errors in one MAi and two MAc patients. Additionally, H19 imprinting errors were observed in elongated spermatids/spermatozoa from one MAi patient. Nevertheless, no statistical differences were found for H19 and MEST global methylation levels (percentage of methylated and unmethylated CpGs, respectively) between patients with complete and incomplete MA and also between MA groups and a control group. These results provide further evidence that imprinting errors occur in spermatogenic cells from patients presenting impaired spermatogenesis, as we and others have previously described in ejaculated and testicular spermatozoa. As paternal imprinting errors can be transmitted to the embryo by the sperm cell, they can provide a possible explanation for poor embryo development and/or low pregnancy rates as correct expression of imprinted genes is crucial for embryo and placental development and function. Therefore, in cases with male factor infertility where unsuccessful in vitro fertilization (IVF) treatments are recurrent, analysis of imprinting marks in spermatozoa might be a useful diagnostic tool.

In vitro exposure to CPF affects bovine sperm epigenetic gene methylation pattern and the ability of sperm to support fertilization and embryo development

“…DNA extraction for each individual sample was performed according to the protocol described by Marques et al (). Briefly, the sperm pellets were overlaid with 100 μL of lysis solution (10 mM Tris–HCl, 25 mM ethylenediaminetetraacetic acid (EDTA), 1% sodium dodecyl sulfate (SDS), 75 mM NaCl) in the presence of 0.2 mg/mL proteinase K, 50 mM DTT and 0.5 μg glycogen (Invitrogen, Cergy‐Pontoise, France) and incubated overnight at 50°C.…”

Epimutations in human sperm from patients with impaired spermatogenesis

“…Moreover, in our studies conducted on testicular sperm, that were individually isolated by micromanipulation from testicular biopsies [6,7], and therefore not contaminated with somatic cells, we have also observed imprinting errors, namely H19 DMR hypomethylation and MEST DMR hypermethylation.…”

Genetic defects in human azoospermia

“…Similarly, a study of the methylation status of the paternally imprinted H19 gene and the maternally imprinted MEST gene in spermatogenic cells from azoospermic patients with either complete or incomplete MA revealed the presence of imprinting errors [182]. Low levels of H19 gene methylation were observed in primary spermatocytes and elongated spermatids, and MEST methylation errors were found in spermatocytes [182].…”

“…For example, it has been suggested that miRNAs (essential for spermatogenesis and possibly involved in the regulation of gene expression) are diagnosis biomarkers for azoospermia. Indeed, miRNA expression was altered in patients, relative to controls [179–182, 184–188]. A recent comparison of men with OA and men with NOA evidenced differences in miRNA expression in spermatogonia, spermatocytes and round spermatids, and thus suggested the presence of epigenetic dysregulation in NOA [189].…”