Triplex Real-time Polymerase Chain Reaction Optimization for AZF Y-chromosome Microdeletion Analysis

Area of Science:

• Genetics
• Reproductive Medicine

Background:

• Y chromosome microdeletions in Azoospermia Factor (AZF) regions (AZFa, AZFb, AZFc) are a significant genetic cause of spermatogenic failure in infertile males.
• No previous studies on AZF microdeletions have been conducted in Ecuador, highlighting a gap in regional genetic diagnostics for male infertility.

Purpose of the Study:

• To optimize a molecular technique for detecting Y chromosome microdeletions in the AZF regions.
• To establish a baseline for AZF microdeletion prevalence in infertile Ecuadorian men.

Main Methods:

• A multiplex real-time quantitative PCR (qPCR) assay was standardized using eight sequence-tagged sites (STSs) and internal controls (SRY, AMELY).
• Genomic DNA from 35 patients with severe oligozoospermia was analyzed using the optimized triplex qPCR assay with EvaGreen dye.

Main Results:

• The optimized triplex qPCR successfully identified microdeletions by analyzing melting temperatures (Tm) of STSs.
• Partial microdeletions in the AZFa region were detected in 8.8% (3 out of 35) of the analyzed patients.
• Specifically, deletions were found in the G34990 and sY85 regions of AZFa; no microdeletions were observed in the AZFb or AZFc regions.

Conclusions:

• The developed triplex qPCR method effectively identifies AZFa, AZFb, and AZFc microdeletions, aiding clinical management of infertile men.
• This study reports the first prevalence data for Ecuador, revealing a higher frequency of AZFa microdeletions compared to other populations.
• The findings underscore the importance of genetic screening for Y chromosome microdeletions in Ecuadorian infertile populations.