Clinical genetic testing

Per guidelines of the American Urological Association, patient samples were tested for common genetic causes of azoospermia⁴. Conventional cytogenetic analysis for affected patients III.3 and III.5 was performed²⁷. Briefly, peripheral blood samples were collected in heparin tubes (BD Biosciences) and were cultured for 72 hours in RPMI-1640 medium supplemented with fetal bovine serum and phytohemagglutinin. Cytogenetic analysis was performed using the GTG banding technique. At least 30 metaphases were analyzed. Genomic DNA was isolated using the Gentra Puregene blood kit (Qiagen). The concentration of DNA was quantified by the Qubit 2.0 fluorometer (Life Technologies), and purity was analyzed by Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). Testing for Y-chromosome microdeletions AZFa, AZFb, and AZFc was performed via fragment analysis for PCR amplified regions sY14, sY86, sY127, and sY254 (Suppl. Table ¹). The PCR products were analyzed on 1.3% agarose gel²⁸. All exons and intron 9 of CFTR were reviewed for CBAVD-associated variants from whole exome sequencing data.