P-079 Transcriptomic Analysis of Seminal Plasma in NOA Men as a Predictor of Successful Testicular Sperm Extraction
Authors: Ng L , Cheung S , Xie P , Rosenwaks Z , Palermo G 
Published: June 2023

Abstract

Study question
Could transcriptomic profiling of semen ejaculate be used to predict successful testicular sperm retrieval for men with idiopathic non-obstructive azoospermia (iNOA)?

Summary answer
Transcriptomic analysis of seminal plasma RNA in iNOA men revealed gene imbalances that consistently correlated with the success of retrieving spermatozoa via a testicular biopsy.

What is known already
Although microdissection testicular sperm extraction (m-TESE) in NOA men results in successful spermatozoa retrieval in about 60% of the cases, it is difficult to predict which cases will fail to recover sperm. While several algorithms have been proposed to make this prediction, such as age, serum FSH, inhibin B, and genetics, there is no one factor that can do so reliably. Even histopathology fails to be more accurate, and it is 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.

Study design, size, duration
Over a 3-year period, 23 men diagnosed with iNOA underwent repeated extensive semen analyses and were deemed azoospermic. These patients were categorized based on whether spermatozoa were subsequently retrieved (+Sperm) or not (-Sperm) at micro-TESE. Transcriptomic analysis was performed by RNAseq, and significant differentially expressed gene (DEG) profiles were assessed and compared between the two cohorts. DNAseq was then used to confirm our findings.

Participants/materials, setting, methods
RNA and DNA were isolated from seminal plasma using a commercially available spin column kit and sequenced by Illumina HiSeq 3000/4000 platform at 2x150bp. Transcriptomic profiling was carried out in comparison to a fertile donor control and among the two cohorts. An absolute log2fold change of &gt; 1 and a P-value of &lt; 0.0005 were considered statistically significant.

Main results and the role of chance
Transcriptomic analyses of ejaculates from 23 azoospermic men were assessed for a total of 21,855 genes against a fertile donor control. Subsequently, 11/23 (47.8%) men (39.0±12yrs) underwent successful testicular sperm retrievals. Their DEG profiles revealed 1,409 imbalanced genes. However, 13 were consistently imbalanced among them: 8 involved in spermatogenesis and 5 in sperm function.
Alternatively, for the 12 men in the –Sperm cohort (34.3±5yrs), 1,265 imbalanced genes were identified with 12 common imbalanced genes associated with spermatogenesis (n = 5), sperm function (n = 3), sperm maturation (n = 1), and cell cycle regulation (n = 3).
When comparing the DEG profiles between the + and –Sperm cohorts, 8 commonly shared imbalanced genes were identified. IGSF11-AS1, a gene expressed in the testis and implicated in spermatid development, was consistently underexpressed in all –Sperm men. TPTE2, a testis-specific gene regulating spermatogenesis, was overexpressed in 81.8% (9/11) of the individuals in the +Sperm cohort and conversely underexpressed in the -Sperm group.
Most interestingly, NEU1, involved in acrosome development and fertilization, was consistently overexpressed in all individuals of the +Sperm group, yet clearly underexpressed in the entire –Sperm group. DNAseq showed that the NEU1 gene exhibited a synonymous mutation for the +Sperm group and a frameshift mutation in the –Sperm group.

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

Wider implications of the findings
Transcriptomics on the ejaculates of men with iNOA represents a noninvasive tool to detect the presence of residual spermatogenesis. Once confirmed, these findings may help guide patients in weighing their exposure to anesthesia and surgical risks. This would help to mitigate patient emotional and financial distress.

Trial registration number
N/A