O-119 Evaluating the reproductive potential of azoospermic men by germline mutation profiling
Authors: Cheung S , Rosenwaks Z , Palermo G D 
Published: August 2021

Study question

Can whole exome sequencing (WES) of spermatozoa from azoospermic men identify mutations related to the etiology of their infertility and ability to support a pregnancy?

Summary answer

Key de novo germline mutations that affect sperm production and/or embryo developmental competence may explain reproductive failure in azoospermic men, regardless of the etiology.

What is known already

Azoospermia accounts for approximately 15% of male factor infertility cases. Although it can be caused by pre-testicular factors, the most recognized forms are testicular and post-testicular. While post-testicular azoospermia is mainly due to a mechanical obstruction, testicular azoospermia, the most severe form, is characterized by scattered functional germinal epithelia that strive to support the meiotic process during gamete development. To shed light on the etiology of this condition, genetic studies have been performed, albeit exclusively on peripheral blood. We chose to perform a genomic assessment of spermatozoa to preferentially detect germline mutations that may be passed to the progeny.

Study design, size, duration

In a 2-year period, we recruited infertile men undergoing epididymal aspiration for acquired obstructive azoospermia (OA; n = 19) or testicular retrieval for nonobstructive azoospermia (NOA; n = 10). Four additional men were included as fertile controls. Following WES, copy number variants (CNVs) and gene mutation profiles were compared between the OA and NOA patients, and within those two categories, in relation to whether they generated a clinical pregnancy (fertile) or not (infertile).

Participants/materials, setting, methods

Spermatozoal DNA was extracted and amplified from the surgically retrieved specimens of consenting men (DNA concentration, 762±492 ng/ul; quality, 1.7±0.1 nm). CNVs, 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 &gt;1.5 or &lt; 0.5 times the median read depth in the control. Common mutations in the OA and NOA cohorts were assessed according to the couples’ clinical outcome.

Main results and the role of chance

Of 29 couples (maternal age, 41.9±7yrs; paternal age, 42.5±7yrs), 19 OA men underwent epididymal sperm retrieval (1.1±4x106/ml concentration, 9±12% motility) while 10 NOA men underwent testicular biopsy (0.03±0.2x106/ml concentration, 0.5±1% motility). WES did not reveal a significant difference in sperm aneuploidy between the two etiologies (OA, 1.8%; NOA, 1.9%).

In OA patients, only 3 genes were deleted, mainly housekeeping-related, while in the NOA cohort, 5 genes were deleted, involved in RNA transcription (POLR2L) and apoptosis (AP5M1), in addition to spermiogenic functions (AP1S2, AP1G2, APOE).

OA patients and their partners (maternal age, 36.8±4yrs) underwent 19 ICSI cycles that resulted in a pregnancy and delivery rate of 47.4% (9/19). Those able to reproduce (n = 9) shared a mutation in ZNF749, a gene affecting only sperm production. The infertile individuals (n = 10) all had a deletion on PRB1, controlling essential DNA replication.

NOA men and their partners (maternal age, 38.2±2yrs) underwent 10 ICSI cycles, yielding a clinical pregnancy rate of 70% (7/10). The fertile men (n = 7) had a concurrent gene deletion involved in stem cell lineage differentiation (MPIG6B). Their infertile counterparts (n = 3) had deleted genes involved in spermato/spermio-genesis (n = 6) and, most importantly, in early embryonic development (MBD5, CCAR1, PMEPA1, POLK, REC9, REPIN1, MAPRE3, and ARL4C).

Limitations, reasons for caution

This is a novel study with limited observations. The presence of housekeeping-related mutations in fertile OA men as well as the DNA replication mutation in infertile OA patients, considering the acquired condition, remains puzzling. Although maternal age was controlled for, confounding factors related to the female partner cannot be excluded.

Wider implications of the findings

Screening men for germline mutations provides valuable information on their ability to reproduce, regardless of the etiology of azoospermia. Genome profiling was able to identify reasons for failed reproductive performance in azoospermic men, particularly those individuals with secretory azoospermia (NOA). Genomic profiling may identify gametes with retained embryo developmental competence.

Trial registration number

n/a