Adverse Effects of 5-Aza-2′-Deoxycytidine on Spermatogenesis Include Reduced Sperm Function and Selective Inhibition of de Novo DNA Methylation

Abstract

The anticancer agent, 5-aza-2Ј-deoxycytidine (5-azaCdR, decitabine), causes DNA hypomethylation and a robust, dosedependent disruption of spermatogenesis. Previously, we have shown that altered testicular histology and reduced sperm production in 5-azaCdR-treated animals is associated with decreased global sperm DNA methylation and an increase in infertility and/or a decreased ability to support preimplantation embryonic development. The goal of this study was to determine potential contributors to 5-azaCdR-mediated infertility including alterations in sperm motility, fertilization ability, early embryo development, and sequence-specific DNA methylation. We find that although 5-azaCdR-treatment adversely affected sperm motility and the survival of sired embryos to the blastocyst stage, the major contributor to infertility was a marked (56 -70%) decrease in fertilization ability. Sperm DNA methylation was investigated using Southern blot, restriction landmark genomic scanning, and quantitative analysis of DNA methylation by real-time polymerase chain reaction. Interestingly, hypomethylation was restricted to genomic loci that have been previously determined to acquire methylation during spermatogenesis, demonstrating that 5-azaCdR selectively inhibits de novo methylation activity. Similar to previous studies, we show that mice that are heterozygous for a nonfunctional Dnmt1 gene are partially protected against the deleterious effects of 5-azaCdR; however, methylation levels are not restored in these mice, suggesting that adverse effects are due to another mechanism(s) in addition to DNA hypomethylation. These results demonstrate that clinically relevant doses of 5-azaCdR specifically impair de novo methylation activity in male germ cells; however, genotype-specific differences in drug responses suggest that adverse reproductive outcomes are mainly mediated by the cytotoxic properties of the drug.

The European Medicines Agency Review of Decitabine (Dacogen) for the Treatment of Adult Patients With Acute Myeloid Leukemia: Summary of the Scientific Assessment of the Committee for Medicinal Products for Human Use

“…No formal carcinogenicity studies were conducted as part of the decitabine development program on the basis of its intended clinical use as an anticancerdrug for treatment ofolder patients with AML who have limited life expectancy; however, evidence from the literature indicates that decitabine has carcinogenic potential. Available data also indicate that decitabine has adverse effects on all aspects of the reproductive cycle, including fertility, embryo-fetal development, and postnatal development [36][37][38][39]. Most toxicities observed from treatment with decitabine are likely to be related to the cytoxicity of the molecule.…”

Semen samples showing an increased rate of spermatozoa with imprinting errors have a negligible effect in the outcome of assisted reproduction techniques

“…The authors suggested that each of the regions present a different vulnerability to present alterations in function of the degree of tandem repetitions that they contain. In mice, different studies have demonstrated that anomalies of the genes that codify for DNMT [32][33][34][35][36][37] or variations in the functionality of these proteins due to interaction with environmental factors 10,[38][39][40] produce methylation alterations in regions regulated by genetic imprinting, causing infertile phenotypes.…”

Routine cryopreservation of spermatozoa is safe — Evidence from the DNA methylation pattern of nine spermatozoa genes

“…During spermatogenesis the genome undergoes massive epigenetic modifications which are crucial for fertilization and embryogenesis [11,17,30]. Early primordial germ cells (PGCs) possess a somatic-like methylation pattern with a high degree of genome-wide DNA methylation.…”