Epigenetic heterogeneity of developmentally important genes in human sperm: Implications for assisted reproduction outcome

<div><p>The molecular basis of male infertility is poorly understood, the majority of cases remaining unsolved. The association of aberrant sperm DNA methylation patterns and compromised semen parameters suggests that disturbances in male germline epigenetic reprogramming contribute to this problem. So far there are only few data on the epigenetic heterogeneity of sperm within a given sample and how to select the best sperm for successful infertility treatment. Limiting dilution bisulfite sequencing of small pools of sperm from fertile donors did not reveal significant differences in the occurrence of abnormal methylation imprints between sperm with and without morphological abnormalities. Intracytoplasmic morphologically selected sperm injection was not associated with an improved epigenetic quality, compared to standard intracytoplasmatic sperm injection. Deep bisulfite sequencing (DBS) of 2 imprinted and 2 pluripotency genes in sperm from men attending a fertility center showed that in both samples with normozoospermia and oligoasthenoteratozoospermia (OAT) the vast majority of sperm alleles was normally (de)methylated and the percentage of epimutations (allele methylation errors) was generally low (<1%). However, DBS allowed one to identify and quantify these rare epimutations with high accuracy. Sperm samples not leading to a pregnancy, in particular in the OAT group, had significantly more epimutations in the paternally methylated <i>GTL2</i> gene than samples leading to a live birth. All 13 normozoospermic and 13 OAT samples leading to a child had <1% <i>GTL2</i> epimutations, whereas one (7%) of 14 normozoospermic and 7 (50%) of 14 OAT samples without pregnancy displayed 1–14% <i>GTL2</i> epimutations.</p></div

Epigenetic dysregulation in the developing Down syndrome cortex

<p>Using Illumina 450K arrays, 1.85% of all analyzed CpG sites were significantly hypermethylated and 0.31% hypomethylated in fetal Down syndrome (DS) cortex throughout the genome. The methylation changes on chromosome 21 appeared to be balanced between hypo- and hyper-methylation, whereas, consistent with prior reports, all other chromosomes showed 3–11 times more hyper- than hypo-methylated sites. Reduced <i>NRSF/REST</i> expression due to upregulation of <i>DYRK1A</i> (on chromosome 21q22.13) and methylation of REST binding sites during early developmental stages may contribute to this genome-wide excess of hypermethylated sites. Upregulation of <i>DNMT3L</i> (on chromosome 21q22.4) could lead to <i>de novo</i> methylation in neuroprogenitors, which then persists in the fetal DS brain where <i>DNMT3A</i> and <i>DNMT3B</i> become downregulated. The vast majority of differentially methylated promoters and genes was hypermethylated in DS and located outside chromosome 21, including the protocadherin gamma (<i>PCDHG</i>) cluster on chromosome 5q31, which is crucial for neural circuit formation in the developing brain. Bisulfite pyrosequencing and targeted RNA sequencing showed that several genes of <i>PCDHG</i> subfamilies A and B are hypermethylated and transcriptionally downregulated in fetal DS cortex. Decreased <i>PCDHG</i> expression is expected to reduce dendrite arborization and growth in cortical neurons. Since constitutive hypermethylation of <i>PCDHG</i> and other genes affects multiple tissues, including blood, it may provide useful biomarkers for DS brain development and pharmacologic targets for therapeutic interventions.</p