P-115 The ageing sperm: molecular mechanisms underlying the age-associated decline in human sperm quality
Authors: Santiago J , Silva J.V , Santos M.A.S , Fardilha M 
Published: June 2022

Abstract

Study question
Are there ageing-related alterations in human sperm protein and small RNA content that can be responsible for the age-associated decline in male fertility?

Summary answer
Our results revealed a set of proteins and small RNAs, particularly miRNAs, that are altered in older men.

What is known already
Male infertility is a common health problem strongly influenced by lifestyle and environment. Advanced paternal age, in particular, has been largely associated with alterations in testicular structure and function, impaired semen parameters and DNA integrity, lower pregnancy rates and decline in offspring fitness. The decline in sperm quality with age was also associated with an increase in oxidative stress. However, only a few studies reported the deregulation of specific sperm proteins or RNAs associated with this risk factor for male infertility.

Study design, size, duration
A hundred and twenty Portuguese men from the Aveiro region were recruited between January 2019 and December 2020 at Hospital Infante D. Pedro E.P.E. (Aveiro, Portugal). All donors provided semen samples for in vitro studies with human spermatozoa. Samples were divided into four groups according to men’s age: (G1) less than 30 years; (G2) between 30 and 35 years; (G3) between 35 and 40 years and (G4) more than 40 years.

Participants/materials, setting, methods
One hundred twenty human sperm samples from volunteer donors were included in this study. Basic semen analyses were performed according to WHO’s guidelines. To avoid the possible contamination by somatic cells, density gradient sperm selection was performed. Nineteen normozoospermic human sperm samples were divided into four groups according to their age and their proteome was evaluated by quantitative proteomic analysis. The small RNA content of sixteen human sperm samples was investigated using small RNA sequencing.

Main results and the role of chance
In this study 120 men aged between 19- and 56-years old (mean age 35.2 ± 6.32 years) were recruited. Our data showed no correlation between paternal age and any seminal parameter investigated, contrary to what was previously described in other study populations. Proteomic analyses revealed 46 differentially expressed proteins (DEPs) between the four study groups (p-value&lt; 0.05;|log2FC|=1.5). In particular, lysosomal protein LAMP1 was significantly upregulated in sperm from men younger than 30 years old compared with men with more than 35 years old. In men younger than 35 years old, cytochrome c oxidase subunit 3 (MT-CO3) and DnaJ homolog subfamily A member 1 (DNAJA1) were consistently downregulated in relation to sperm from men aged between 35 and 40 years old. Gene ontology analysis of all the deregulated sperm proteins shown that response to unfolded protein, positive regulation of mitochondrion organization, negative regulation of phosphoprotein phosphatase activity, positive regulation of apoptotic process, and spermatogenesis are common biological processes affected. Transcriptomic analysis identified 5 differentially expressed miRNAs (DEMs) between the four groups studied (p-value&lt; 0.05), among which has-miR-374c-3p and miR-103a-3p were significantly upregulated in men younger than 35 years old compared to sperm from men aged between 35 and 40 years old.

Limitations, reasons for caution
The major limitations of this study are the relatively small sample size and the limited number of participants younger than 20 years and older than 45 years. Additionally, we cannot exclude the influence of unmeasured confounders, including lifestyle factors such as alcohol consumption, diet, exercise, and stress, in our findings.

Wider implications of the findings
Despite reproductive history and basic semen analysis being the primary steps in the assessment of male infertility, this routine examination is insufficient to explain almost 30% of the cases. The DEPs and DEMs identified could help to elucidate and/or became potential diagnostic markers for age-associated decline in human sperm quality.

Trial registration number
not applicable