Investigation of DAZ and RBMY1 gene expression in human testis by quantitative real-time PCR

Area of Science:

• Reproductive Biology
• Human Genetics
• Molecular Diagnostics

Background:

• Male azoospermia is a complex condition often associated with genetic factors affecting spermatogenesis.
• The DAZ (Deleted in Azoospermia) and RBMY1 (RNA-binding motif protein Y1) genes are crucial for male fertility and are located on the Y chromosome.
• Y-chromosome microdeletions, particularly in the AZFc and AZFb regions, are known causes of severe spermatogenic failure.

Purpose of the Study:

• To develop and validate quantitative real-time PCR assays for assessing the copy number of DAZ and RBMY1 genes in testicular tissue.
• To investigate the correlation between DAZ and RBMY1 gene copy numbers and the histopathological findings in azoospermic males.
• To evaluate the predictive value of these assays for sperm recovery in patients undergoing testicular sperm extraction (TESE).

Main Methods:

• Quantitative real-time PCR (qPCR) assays were developed for the DAZ and RBMY1 genes.
• RNA was extracted from testicular biopsies of normospermic controls (n=6) and azoospermic males (n=17).
• Histopathological analysis of testicular biopsies was performed, and Y-chromosome microdeletions were identified in two azoospermic patients.

Main Results:

• A significant reduction in the copy number of at least one gene (DAZ or RBMY1) was observed in 40% of the azoospermic cohort (DAZ P=0.003; RBMY1 P=0.009).
• Histopathology in affected patients ranged from Sertoli cell only (SCO) to severe hypospermatogenesis with fibrosis.
• The patient with AZFb+c deletion showed no DAZ/RBMY1 expression (SCO), while the AZFc deletion patient had reduced RBMY1 and no DAZ expression (spermatocyte arrest).

Conclusions:

• Reduced copy numbers of DAZ and RBMY1 genes are significantly associated with azoospermia and specific histopathological findings.
• The qPCR assays demonstrated positive predictive values of 78% for DAZ and 70% for RBMY1 in predicting sperm recovery via TESE.
• These genetic assays offer valuable diagnostic tools for male infertility, aiding in the assessment of spermatogenic potential and guiding assisted reproductive technology decisions.