THE EFFECT OF GERMLINE MUTATIONS ON THE ABILITY OF SURGICALLY RETRIEVED SPERMATOZOA FROM AZOOSPERMIC MEN TO SUPPORT EMBRYONIC DEVELOPMENT

Abstract

To identify eventual germline mutations involved in spermatogenesis and the ability of spermatozoa retrieved from azoospermic men to support embryonic development. For over 20 months, we recruited infertile men undergoing epididymal aspiration for obstructive (OA; n=17) and testicular retrieval for nonobstructive (NOA; n=10) origins. Three men functioned as fertile controls. Gene mutations were compared according to the etiology of the azoospermia and in relation to whether they were able to generate a pregnancy (fertile) or not (infertile). Specimens were provided by consenting men, who were being treated at our center for infertility. DNA was extracted and amplified from at least 500 spermatozoa (DNA concentration, 705±562 ng/ul; quality, 1.7±0.1 nm). Following NGS, gene mutations, duplications, and deletions were detected using the CLC Genomic Server 9.0. Genes were considered duplicated or deleted when the read depth was >1.5 or <0.5 times the median read depth in the control. Common gene mutations in the OA and NOA cohorts were assessed according to the couples’ clinical outcomes. Of the 27 couples (paternal age, 41.3±5yrs) included, 17 OA men underwent epididymal sperm retrieval with an average concentration of 1.3±3x106/ml and 7±14% motility, while 10 NOA men yielded twitching spermatozoa with a concentration of 0.03±0.2x106/ml and 0.5±1% motility. Whole genome karyotype assessment showed an overall sperm aneuploidy rate below only 2%, specifically 1.7% in the OA and 1.9% in the NOA cohort. In the OA group overall, only 3 housekeeping genes were mutated (ATP4A, SLC17A7, and OR1D4). However, in the NOA cohort, 5 genes involved in RNA transcription (POLR2L), apoptosis (AP5M1), and basic spermiogenic function (AP1S2, AP1G2, and APOE) were deleted. The OA men and their partners (maternal age, 34.8±3yrs) underwent 17 ICSI cycles, resulting in a pregnancy and delivery rate of 47.1% (8/17). For those able to reproduce (n=8), all shared a mutation in ZNF749, which is mainly responsible for spermatogenesis. Those who remained infertile had a common mutation in PRB1, a gene involved in DNA replication. When NOA men were treated in 10 ICSI cycles with their partners (maternal age, 38.2±2yrs), the pregnancy rate was 70% (7/10). Of those 7 men, 1 mutation (MPIG6B) related to stem cell lineage differentiation was in common. The remaining 6 who remained childless had mutations in genes associated with spermatogenesis (6 genes), apoptosis (4 genes), acrosomal function (2 genes), and, most importantly, in early embryonic development (MBD5, CCAR1, PMEPA1, POLK, REC9, REPIN1, MAPRE3, and ARL4C). Screening infertile men for germline mutations can help characterize spermatogenic function in relation to the etiology of their azoospermia and provide valuable information on their reproductive potential. In men capable of reproducing, gene mutations were limited to those associated with spermatogenic function. For men unable to reproduce, particularly the NOA cohort, the mutated genes were mostly related to multiorgan functions and embryo development.