P-029 Genome-wide microRNA expression profiling in human spermatozoa and its relation to sperm quality
Authors: Cassuto N  G , Ruoso L , Keromnes G , Prat-Ellenberg L , Ledee N , Chao C , Mouik H , Assou S 
Published: July 2024

Abstract

Study question
Does the microRNA (miRNA) expression profile in human spermatozoa change with their morphological appearance?

Summary answer
This study revealed miRNA expression associated with good-quality sperm classified based on their morphology. These miRNAs could represent efficient biomarkers to evaluate male fertility status.

What is known already
We previously published a scoring scale where the spermatozoa with a Score 6 (Good spermatozoon) display normal head shape with symmetrical nuclear no extrusion and/or no invagination of the nuclear membrane, without any vacuole and normal base. The spermatozoa with a Score 0, display a nuclear-shape disorder with an abnormal base and at least one large vacuole (Bad spermatozoon). Despite the great magnitude of studies investigating the miRNA role in spermatogenesis, limited work has been done to evaluate it as a potential biomarker for sperm quality in relation to their morphology.

Study design, size, duration
In a prospective study from March 2023 to May 2023, twenty semen samples were collected. The samples were categorized according to their scoring morphology into Score 6 and Score 0. Ten samples (five with a Score 6 and five with a Score 0) were used for the analysis by microarray and ten (five with a Score 6, and five with a Score 0) for the miRNA expression analysis by RT-qPCR.

Participants/materials, setting, methods
Semen samples were scored according to their high-magnification morphology and stored at -80 °C. We used miRNA microarray chip to analyze miRNA profile. Statistical significance was evaluated by Significance Analysis of Microarrays, screened by Fold change&gt;2 and p-values ≤ 0.05. The correlation between miRNAs and their corresponding mRNA targets was analyzed using in silico prediction algorithms. The methylome was studied using the DNA sequencing method. TaqMan miRNA assays were used to confirm the miRNA results.

Main results and the role of chance
We perform a robust transcriptomic analysis to define unique miRNA signatures able to identify the spermatozoa that have high scores regardless of morphological appearance at high magnification. There were 86 significantly overexpressed in Score 6 and 21 miRNAs in Score 0. Score 6-spermatozoa had significantly higher levels of hsa-miR-132 (FCx41, p=1.68E-8), hsa-miR-1973 (FCx32, p=6.19E-8), hsa-miR-34 (FCx30, p=8.43E-8), hsa-miR-25 (FCx27, p=1.30E-6), hsa-miR-342 (FCx24, p=1.57E-5), hsa-miR-15 (FCx14, p=8.56E-6), hsa-miR-125 (FCx13, p=6E-4), hsa-miR-30 (FCx12, p = 3.75E-6), than Score 0. The miRNAs overexpressed in Score 6 are predicted to target 910 messenger RNA involved in biological functions of the spermatozoa and several signaling pathways, such as mTOR, p53, and MAPK signaling pathways, as well as cell Intrinsic pathway for apoptosis, cellular senescence, and oxidative stress. Whereas genes targeted by score 0-miRNAs were implicated in the regulation of cellular growth and proliferation. Our data indicate a difference in methylation of the promoter region of miRNAs, and their validated target genes were observed between score 6 and score 0. Aberrant expression of the Score 6-miRs or their targets could be due to the methylation status of the promoter region and indicative of a defect in spermatogenesis.

Limitations, reasons for caution
This work provides a foundation for the interpretation of miRNA changes associated with spermatozoa morphological appearance. The data must be validated by TaqMan miRNA assays in a higher number of semen samples. Moreover, the molecular network of miRNA-targeted gene pathways also merits investigation. These experiments are currently ongoing.

Wider implications of the findings
The profiling of miRNAs repertoire in scored spermatozoa morphology opens new perspectives to explore male fertility status and provides a biomarker panel for sperm analysis during the ART procedure. Larger well-controlled investigations employing microRNAome approaches are needed to validate these conclusions.

Trial registration number
not applicable