Triplex Real-time Polymerase Chain Reaction Optimization for AZF Y-chromosome Microdeletion Analysis
Tatiana Puga Torres1, Xavier Blum Rojas1, Medardo Blum Narváez1
1Centro Nacional de Reproducción Asistida INNAIFEST - Guayaquil - Ecuador.
Objective:
Y chromosome microdeletions at the "Azoospermia Factor" regions (AZFa, AZFb, AZFc) are the second genetic cause of spermatogenic failure in infertile men. Despite its importance for the treatment of infertile patients, no prior investigations have been previously published in Ecuador. . The purpose of this study is to optimize a molecular technique that allows detection of microdeletions in the AZF region.
Methods:
Using a genomic DNA of healthy male with natural conceived offsprings, a multiplex real time polymerase chain reaction (qPCR) was standarized with eigth sequence-tagged site (STS) sY85, G34990, sY133, sY127, sY254, sY255, and using as internal control sex-determine region Y (SRY) and Ameologenin Y (AMELY). With this technique, 35 DNA samples taken from peripheral blood of patients with severe oligozoospermia were analyzed.
Results:
A triplex qPCR was standardized using EvaGreen DNA-binding dye to obtain melting temperature (Tm) of the STS previously mentioned. Three of the patients evaluated were detected to have partial microdeletion in the AZFa region, with a frequency of 8.8%; being losses in the G34990 section (one patient) and sY85 section (two patients). No cases of microdeletions in other AZF regions were found.
Conclusion:
The triplex qPCR optimizated allows the identification of microdeletions in AZFa, AZFb and AZFc region in infertile men and a better clinical management of the patient's treatment decision. This first report for Ecuador reveled a higher prevalence of microdeletions in the AZFa region in comparison with those previously described in other populations.
