Dysregulation of the human B-defensin 128 gene impacts sperm function

OBJECTIVE:
Male infertility is a global issue and many of the cases of male infertility have unknown causes. Genetic mutations may be a causative factor in a significant number of idiopathic cases. We conducted Whole Exome Sequencing (WES) analysis in men undergoing assisted conception and identified a genetic mutation in the β-defensin 128 (DEFB128) gene. The DEFB128 gene is explicitly expressed in the male reproductive tract, where it forms part of the sperm glycocalyx and protects sperm cells in the female reproductive tract.

MATERIALS AND METHODS:
DNA was extracted from the blood of 26 men attending the assisted conception unit and sequenced using WES. A pathogenic variant in the β-defensin 128 gene was identified using bioinformatics analysis and validated using Sanger sequencing. Enzyme-linked immunosorbent assay (ELISA) was used to detect the presence of the DEFB128 protein on sperm cells. wtDEFB128 recombinant protein was brought commercially and anti-microbial assays against a range of control strain Gram-positive and Gram-negative bacteria commonly found in the female reproductive tract were performed.

RESULTS:
Based on bioinformatics analysis, we identified a novel pathogenic homozygous frame-shift insertion causing a premature stop codon in the DEFB128 gene from one patient. Sanger sequencing was used to confirm the pathogenic mutation in the patient. DEFB128 has been shown in literature to be highly and exclusively expressed in the male tissue, presence of DEFB128 on sperm was investigated using ELISA assays. In order to identify the functional significance of this protein, antimicrobial assays were performed using the commercially available DEFB128 recombinant protein.

Based on our results, we hypothesise that the loss of the DEFB128 protein reduced the competence of the patients sperm cells by making them more prone to infections in the female reproductive tract.

CONCLUSIONS:
The identified DEFB128 mutation (rs11396059) has not been associated with any disease to date. Reduced antimicrobial activity due to the pathogenic mutation may render sperm cells with less protection against pathogens in the female reproductive tract. As the patient with the DEFB128 mutation has no other medical condition we predict that it is the cause of infertility. However, the exact physiological impairment in-vivo needs to be confirmed.

Furthermore, like the human DEFB126 protein, the DEFB128 may contribute to the formation of the sperm glycocalyx and hence it may be involved in the efficient movement of sperm in the female reproductive tract. Sperm penetration assays will need to be performed on affected patients sperm cells to provide further evidence of functional failure.

IMPACT STATEMENT:
Many cases of idiopathic male infertility remain a mystery. This may be in-part due to the lack of understanding of the physiological regulation of sperm cells. The DEFB128 gene is exclusive expressed in the male reproductive tract and forms part of the sperm glycocalyx. Understanding the sperm glycocalyx and its functional significance will help in understanding the cause of dysfunction in many cases of idiopathic male infertility.