To query genes involved in spermatogenic function and embryonic competence of spermatozoa isolated from azoospermic men. NGS assessment was performed on surgically retrieved spermatozoa from 9 azoospermic men categorized as obstructive (OA) or non-obstructive (NOA) based on their spermatogenetic profile. Gene mutations and ICSI pregnancy outcomes were assessed and compared between the two groups. Specimens were provided by consenting men being treated at our center for infertility. DNA extraction was performed from at least 500 spermatozoa, followed by PCR-based random hexamer amplification (DNA concentration, 395±217ng/ul; quality, 1.7±0.1nm). Following NGS, gene mutations, including duplications and deletions, were detected by CASAVA and VarScan2 software programs. Our analysis included a total of 9 men with an average age of 45.2±8yrs. Six OA patients (average concentration of 2.0±3x106/ml and motility of 0.5±1%) were treated by epididymal aspiration or testicular biopsy. In all OA cases, obstruction was due to a prior vasectomy. These men underwent 6 cycles, with a pregnancy and delivery rate of 50%. Three NOA men underwent testicular biopsies that yielded spermatozoa in all cases. They were treated by ICSI in 3 cycles, with a pregnancy and delivery rate of 66.7%. NGS showed no differences in the aneuploidy of spermatozoa from the OA and NOA groups. DNA sequencing was performed on 70,000 genes, of which only two were mutated in the OA patients, while NOA men had 123 genes mutated (P<0.0001). Among these genes, the most represented were related to mRNA transcription (n=26), spermatogenesis (n=15), DNA repair (n=14), centrosomal function (n=13), and apoptosis (n=12). When we further attempted to identify the gene mutation profile of the OA men in relation to their ability to generate pregnancy, we found a mutation of MGAM in the fertile group, while ARHGEF5 and PRB1 were mutated in the infertile group. These mutations were unrelated to spermatogenesis or embryo development, as expected. When a similar assessment was carried out in the NOA group, the infertile men had mutations in 6 genes related to early embryonic development (IFRD2, HYAL1, INSM1, ECSIT, RYR1, and MFNG). For the fertile men, in addition to 4 gene mutations related to cell apoptosis, 6 more mutations were involved in organ development. Screening infertile men for gene mutations helps characterize the spermatogenic function of their germinal epithelium. Men with secretory azoospermia have a mutated set of genes with compromised function responsible for the impaired meiotic process. OA men did not have definite gene mutations related to their ability to achieve a pregnancy. Infertile NOA men had mutated genes responsible for early embryonic growth, whereas their fertile counterparts had deleted genes involved in organ development. This may explain the higher incidence of neonatal malformation observed with severe male factor infertility.