O-259 Seminal plasma cell-free RNA to predict testicular biopsy sperm retrieval outcome in azoospermic men
Authors: Ng L , Cheung S , Xie P , Rosenwaks Z , Palermo G  D 
Published: July 2024

Abstract

Study question
Is there a way to assess seminal plasma of NOA men to predict a successful sperm retrieval at testicular biopsy?

Summary answer
Multi-omics approach in seminal plasma of NOA men provides reliable prediction on the spermatogenic activity within the seminiferous tubule.

What is known already
Microdissection testicular sperm extraction (m-TESE) in NOA men successfully retrieved spermatozoa in about 60% of the cases. While several algorithms have been investigated aiming at estimating spermatogenic progress such as age, serum FSH, inhibin B, and genetics, there is no specific assay that can do so reliably. Even histopathology is often inconclusive while equally invasive. Recently, epigenetic analysis on testicular biopsy specimens has shown a differential gene expression in relation to the origin of azoospermia and spermatogenic function (Cheung, et al, 2024, in press).

Study design, size, duration
Over a 4-year period, twenty-eight men were deemed azoospermic following several failed extensive semen analyses. Transcriptomic analysis was performed by RNAseq on seminal fluid and the specific gene expression were evaluated and compared. In addition, DNAseq and proteomics were carried out to confirm our findings.
These patients then underwent micro-TESE by a single surgeon and were divided into two cohorts based on whether spermatozoa was successfully retrieved or not.

Participants/materials, setting, methods
RNA and DNA were isolated from seminal plasma using a commercial kit and sequenced by Illumina HiSeq3000/4000 platform. Differentially expressed genes (DEGs) were assessed and compared to a fertile donor control using DESeq2 and Gene Ontology analysis. An absolute log2fold change of &gt; 1 and a P-value of &lt; 0.0005 were considered statistically significant. Gene mutations were detected by CLC Genomic Server 9.0.

Main results and the role of chance
Transcriptomic in 28 ejaculates from iNOA men browsed 21,855 genes against a fertile donor. Of 3,505 imbalanced genes, 12 were consistently imbalanced in 12 men, while 19 genes were imbalanced in remaining 16. Among the 2 cohorts, 8 common imbalanced genes were found. Particularly TPTE2, a testis-specific gene that regulates spermatogenesis, was overexpressed in 10/12 men (OE-12-cohort), while underexpressed in the remainder 16 (UE-16-cohort). DNAseq showed that TPTE2 was conserved in the OE-12-cohort, while exhibiting a frameshift mutation in the UE-16-cohort.
Most interestingly, NEU1, a gene involved in acrosome development and fertilization, was found to be consistently and concurrently overexpressed in the OE-12-cohort, while being convserly underexpressed in all the men in the UE-16 cohort. DNAseq confirmed the NEU1 gene exhibited a synonymous mutation in the OE-12 and a frameshift mutation in the UE-16-cohort.
About 1-2 months after the DEG analysis was completed, all of the patients underwent m-TESE. The 12 men from the OE-12-cohort all had successful retrieval of spermatozoa at m-TESE, conversely the 16 men from the UE-16-cohort all consistently failed to yield spermatozoa at testicular surgery in spite of extensive search for several hours and by multiple embryologists. Moreover, histopathology confirmed spermatogenic status in both OE-12&UE-16-cohorts.

Limitations, reasons for caution
Using non-invasive RNAseq and DNAseq on the seminal plasma has allowed us to identify DEGs that may be used to predict whether a patient with iNOA would have a successful or failed sperm retrieval with micro-TESE. However, these results are preliminary and should be further validated in larger study cohort.

Wider implications of the findings
Transcriptomics on the ejaculates of men with iNOA represents a noninvasive tool to detect presence of residual spermatogenesis. If confirmed by further studies, this precision medicine method may be utilized in order to properly counsel patients regarding their future prognosis.

Trial registration number
Not applicable