# D4Z4 Hypomethylation in Human Germ Cells

## TL;DR

This study investigates D4Z4 methylation in human germ cells and finds that most sperm cells are unmethylated, while immature oocytes show higher methylation.

## Contribution

The study provides new insights into D4Z4 methylation patterns in human germ cells and their potential impact on assisted reproduction outcomes.

## Key findings

- Sperm samples showed an average D4Z4 methylation of 2.5%, with 78% of sperm cells completely unmethylated.
- Only 17.5% of immature oocytes had D4Z4 methylation below 2.5%, while many showed hypermethylation.
- Lower D4Z4 methylation in sperm and oocytes was associated with higher pregnancy rates in assisted reproduction.

## Abstract

Expression of the double homeobox 4 (DUX4) transcription factor is highly regulated in early embryogenesis and is subsequently epigenetically silenced. Ectopic expression of DUX4 due to hypomethylation of the D4Z4 repeat array on permissive chromosome 4q35 alleles is associated with facioscapulohumeral muscular dystrophy (FSHD). In peripheral blood samples from 188 healthy individuals, D4Z4 methylation was highly variable, ranging from 19% to 76%, and was not affected by age. In 48 FSHD2 patients, D4Z4 methylation varied from 3% to 30%. Given that DUX4 is one of the earliest transcribed genes after fertilization, the D4Z4 array is expected to be unmethylated in mature germ cells. Deep bisulfite sequencing of 188 mainly normozoospermic sperm samples revealed an average methylation of 2.5% (range 0.3–22%). Overall, the vast majority (78%) of individual sperm cells displayed no methylation at all. In contrast, only 19 (17.5%) of 109 individual germinal vesicle (GV) oocytes displayed D4Z4 methylation <2.5%. However, it is not unexpected that immature GV oocytes which are not usable for assisted reproduction are endowed with D4Z4 (up to 74%) hypermethylation and/or abnormal (PEG3 and GTL2) imprints. Although not significant, it is interesting to note that the pregnancy rate after assisted reproduction was higher for donors of sperm samples and oocytes with <2.5% methylation.