O-311 Evaluating the Reproductive Potential of Azoospermic Men by Profiling the Genome of Surgically Retrieved Spermatozoa
Authors: Chung P , Cheung S , Rosenwaks Z , Palermo G.D 
Published: June 2022

Study question

Can whole exome sequencing of surgically retrieved spermatozoa from azoospermic men pinpoint mutations related to the etiology of their infertility and ability to support pregnancy?

Summary answer

Identifying key germline mutations involved in spermatogenesis helps explain reproductive failure, regardless of the etiology of the azoospermia.

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. Post-testicular azoospermia is mainly attributed to a mechanical obstruction, whereas testicular azoospermia, the most challenging form, is characterized by scattered functional germinal epithelia that may fail to support the meiotic process during sperm development. To elucidate the etiology of this condition, genetic studies on somatic cells have been carried out. Here, we perform whole exome sequencing (WES) on surgically retrieved spermatozoa to preferentially detect germline mutations that may be passed on to offspring.

Study design, size, duration

Over 3 years, we recruited patients undergoing epididymal sperm aspiration for acquired obstructive azoospermia (OA; n = 19) or testicular biopsy for nonobstructive azoospermia (NOA; n = 11). Eight men were included as fertile controls. Copy number variants (CNVs) and gene mutation profiles were obtained through WES 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.

Participants/materials, setting, methods

Spermatozoal DNA was extracted and amplified from the surgically retrieved specimens (concentration, 742±520 ng/ul; quality, 1.7±0.1 nm). CNVs and gene mutations were detected using 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 were compared between the OA and NOA cohorts, as well as according to the couples’ clinical outcomes. Female partners had negative infertility workups.

Main results and the role of chance

Of 30 men (paternal age, 42.3±7yrs), 19 OA men underwent epididymal sperm retrievals (concentration, 1.1±4x106/ml, 9±12% motility), while 11 NOA men underwent testicular biopsies (concentration, 0.03±0.4x106/ml, 0.5±1% motility). WES did not indicate a significant difference in sperm aneuploidy between the two etiologies (OA, 1.7%; NOA, 1.8%) compared to the control (1.1%).

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

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

NOA men and their partners (maternal age, 38.2±2yrs) underwent 11 ICSI cycles, yielding a delivery rate of 72.7% (8/11). The fertile men (n = 8) all shared a MPIG6B deletion, involved in stem cell lineage differentiation. All of their infertile counterparts (n = 3) presented gene deletions not only involved in spermato/spermio-genesis (n = 4) but, most importantly, also superimposed with those encoding early embryonic development (MBD5, CCAR1, PMEPA1, POLK, REC8, REPIN1, MAPRE3, ARL4C).  

Limitations, reasons for caution

Although maternal age was controlled for, confounding factors related to the female partner cannot entirely be excluded. While men unable to reproduce shared common gene mutations providing information about their condition, these findings still need to be confirmed in larger observations.

Wider implications of the findings

By performing WES, we were able to identify specific mutations associated with compromised embryo developmental competence of surgically retrieved spermatozoa. DNA sequencing technologies help identify gametes capable of sustaining a pregnancy even in the most severe form of male infertility, laying the groundwork for precision medicine in this field.

Trial registration number

N/A