GENOMIC PROFILING OF SURGICALLY RETRIEVED SPERMATOZOA TO EVALUATE THE REPRODUCTIVE POTENTIAL OF AZOOSPERMIC MEN

Abstract

To identify, by whole exome sequencing (WES), gene mutations affecting the embryo developmental competence of spermatozoa from azoospermic men. Over 2 years, we recruited patients undergoing epididymal sperm aspiration for acquired obstructive azoospermia (OA; n=19) or testicular retrieval for nonobstructive azoospermia (NOA; n=11). Eight additional men were included as fertile controls. Spermatozoal DNA was extracted and amplified from the surgically retrieved specimens (concentration, 742±520 ng/ul; quality, 1.7±0.1 nm). Copy number variants (CNVs) and gene mutations were detected using CLC Genomic Server 9.0 and compared between the OA and NOA cohorts, followed by sub-analyses within those two categories according to whether they generated a clinical pregnancy (fertile) or not (infertile), while controlling for maternal age. Of 30 men (paternal age, 42.3±7yrs), 19 OA men underwent epididymal sperm retrieval (1.1±4x106/ml concentration, 9±12% motility), while 11 NOA men underwent testicular biopsy (0.03±0.4x106/ml concentration, 0.5±1% motility). WES did not reveal a significant difference in sperm aneuploidy between the two etiologies (OA, 1.7%; NOA, 1.8%) compared to the control (1.1%). When assessing the origin of azoospermia, we found that the OA group had only 3 housekeeping genes deleted, while 5 genes in the NOA cohort were deleted; these 5 are involved in RNA transcription (POLR2L), apoptosis (AP5M1), and spermiogenic functions (AP1S2, AP1G2, APOE). We then assessed the reproductive potential of these men. The OA group underwent 19 ICSI cycles with their female partners (maternal age, 36.8±4yrs), resulting in a clinical pregnancy/delivery rate of 47.4% (9/19). Of the couples who delivered (n=9), all the men shared a mutation only in ZNF749, a transcriptional regulation gene. Of those OA men who remained infertile (n=10), all had a deletion on PRB1, which is associated with controlling essential DNA replication. When we assessed the NOA men who underwent 11 ICSI cycles with their female partners (maternal age, 38.2±2yrs), yielding a delivery rate of 72.7% (8/11), the fertile men (n=8) had only a gene deletion involved in stem cell lineage differentiation (MPIG6B) in common. However, their infertile counterparts (n=3) all had commonly deleted genes not only involved in spermato/spermio-genesis (n=6) but, most importantly, also associated with encoding early embryonic development (MBD5, CCAR1, PMEPA1, POLK, REC8, REPIN1, MAPRE3, ARL4C). DNA sequencing of the male gamete can identify germline mutations associated with azoospermia. For individuals capable of reproducing, we found mutations that were mainly limited to spermatogenic function. However, for men unable to sustain a pregnancy, particularly in the NOA cohort, we found gene mutations related to impaired embryo development.