P-712 Transcriptomic Prediction of Spermatogenesis in the Ejaculates of Nonobstructive Azoospermic Men and Genomic Characterization of their Embryo Developmental Competence
Authors: Cheung S , Ng L , Rosenwaks Z , Palermo G D 
Published: June 2023

Abstract

Study question
Can the genomic assessment of ejaculates from azoospermic men predict successful sperm extraction at testicular biopsy (TESE) and provide information on their reproductive potential?

Summary answer
RNAseq on the ejaculates of NOA men identified transcriptomic predictors of successful TESE outcome, while DNAseq evidenced gene-related mechanistics linked to reproductive performance.

What is known already
The most puzzling form of azoospermia is the testicular type. The chances of successful surgical sperm retrieval for these cases can be unpredictable, even when a benign histopathology has been obtained. The ability to identify individuals with complete loss of spermatogenesis at the seminiferous tubule level would help eliminate anesthesia and surgical risks, as well as financial and emotional distress. We propose to noninvasively assess the ejaculates from these men to predict successful TESE sperm retrieval. We utilize DNAseq to assess the impact of germline mutations on the embryo developmental competence of the surgically retrieved gametes.

Study design, size, duration
Over 4 years, we recruited men with normal peripheral karyotypes and azoospermia (n = 23), confirmed by absence of spermatozoa in extensive semen analyses. We performed epigenetic assessments on their ejaculates to compare transcriptomic profiles according to whether spermatozoa were successfully retrieved after TESE ((+)Sperm,n=11), or not ((-)Sperm,n=12). We then performed DNAseq (IRB 1006101085) on the testicular spermatozoa retrieved, to assess their ability to generate a pregnancy (fertile) or not (infertile), while controlling for maternal age.

Participants/materials, setting, methods
RNA was isolated from seminal fluid for epigenetic assessment (21,855 genes). A &gt; 1 absolute log2fold change and P&lt;0.0005 were considered significant. Transcriptomic profiles were compared between the (-)Sperm and (+)Sperm cohorts. For genetic assessment, surgically retrieved spermatozoal DNA from the (+)Sperm and obstructive azoospermic (OA) control (n = 19) cohorts were extracted and amplified. Gene mutation profiles, detected by CLC Genomic Server 9.0, were assessed for the (+)Sperm cohort according to ICSI outcomes, compared to the OA-control.

Main results and the role of chance
Transcriptomic analyses on seminal plasma from the 23 NOA men identified 8 imbalanced genes, involved in spermatogenesis (TMCO5B,C10orf62,SMKR1,SPZ1), sperm function (NEU1,TPTE2), and testis development (TRPC1,IGSF11-AS1). TPTE2 was almost completely expressed in 81.8%(9/11) of the (+)Sperm cohort (paternal age, 37.1±6yrs), while IGSF11-AS1 was underexpressed in all 12 (-)Sperm men (paternal age, 34.3±5yrs). Both genes are implicated in spermatogenic defects and are normally highly expressed in testis. Interestingly, NEU1, localized on the sperm acrosome and crucial for sperm capacitation, was exclusively underexpressed in (-)Sperm while overexpressed in (+)Sperm cohorts.
Genomic analyses of spermatozoal DNA from the (+)Sperm cohort was compared to a matched control of OA patients (paternal age, 36.8±7yrs). Spermatozoa from the study cohort displayed mutations on genes involved in RNA transcription (POLR2L), apoptosis (AP5M1), and spermiogenic functions (AP1S2,AP1G2,APOE), while these genes were unaffected in the OA-control.
When we compared sperm genetic profiles of the successful TESE cohort (maternal age, 36.8±2yrs, paternal age, 37.1±6yrs) according to ICSI outcome, we found that spermatozoa from the 8 fertile patients, all with term pregnancies, displayed consistent mild mutations on MPIG6B (stem cell lineage differentiation) while gametes from their 3 infertile counterparts carried severe mutations on genes (MBD5,CCAR1,PMEPA1,POLK,REC8,REPIN1,MAPRE3,ARL4C) involved in early embryonic development, and therefore failed to achieve embryo implantation.

Limitations, reasons for caution
Although we predicted, by transcriptomic analyses of ejaculates from NOA men, the presence of spermatozoa and characterized their embryo developmental competence through genetic profiling, these preliminary results need validation in a larger study cohort. Moreover, although controlled for, a confounding female factor cannot be undoubtfully excluded.

Wider implications of the findings
Epigenetic assessment of seminal fluid from NOA men can noninvasively identify biomarkers capable of predicting complete loss of spermatogenesis, sparing these patients from unnecessary surgery. Gene profiling can identify gametes capable of supporting term pregnancies, laying the groundwork for precision medicine in male infertility screening.

Trial registration number
n/a