Advanced Paternal Age and Sperm Proteome Dynamics: A Possible Explanation for Age-Associated Male Fertility Decline

Abstract

Male fertility is strongly influenced by environmental exposures, lifestyle, and advancing age. While advanced paternal age (APA) has been linked with a progressive decline in male fertility, poor reproductive outcomes, and decreased offspring health, the molecular mechanisms underlying these alterations remain unclear. In this work, we investigated the impact of men's age on human sperm protein expression and phosphorylation to identify molecular alterations possibly responsible for the age-associated decline in male fertility. Semen samples from volunteers attending fertility consultations at the Hospital of Aveiro were collected, analyzed according to WHO's guidelines, and processed by the density gradient technique. The proteome and phosphoproteome of 19 normozoospermic human sperm samples divided into four age groups were evaluated by mass spectrometry: ≤30 years old; 31-35 years old; 36-40 years old; and >40 years old. Proteomic analysis revealed 46 differentially expressed proteins (DEPs) between groups, some of them associated with infertility-related phenotypes. Gene ontology (GO) analysis, performed using the DAVID database, revealed that DEPs in older men were enriched in pathways related to stress response, metabolism, and embryo implantation. Additionally, 94 differentially phosphorylated sites corresponding to 76 differentially expressed phosphorylated proteins between the groups were identified, related to key reproductive processes such as sperm motility, spermatogenesis, and sperm binding to zona pellucida, and involved in metabolic and stress response pathways, like HSF1 activation. The set of proteins and phosphorylated residues altered in the sperm fraction usually used in assisted reproductive technology (ART) highlights the need to consider the age of the male partner during fertility assessment and treatment planning. These markers can also be used to explain cases of idiopathic infertility, failure in ART, or repeated abortion associated with APA, overcoming the subjectivity of the conventional semen analysis.

Keywords: advanced paternal age; male infertility; phosphorylation; protein; spermatozoa.

Figure 1

The differentially expressed proteins (DEPs) identified between the age groups. (A) Venn diagram displaying the number of DEPs in each group. (B) GO enrichment analyses for the DEPs identified in each age group showed significant (FDR < 0.05) biological processes, molecular functions, and pathways. Age groups: G1, ≤30 years old; G2, 31–35 years old; G3, 36–40 years old; G4, >40 years old.

Figure 2

Identification of global phosphorylated proteins and their phosphorylated sites. (A) Number and distribution of phosphorylation sites of phosphorylated proteins identified. (B) The phosphorylation site distribution of serine (S), tyrosine (Y), and threonine (T). Venn diagram showing the number of common and exclusive differentially expressed phosphorylation sites (C) and proteins (D) between age groups (G1, ≤30 years old; G2, 31–35 years old; G3, 36–40 years old; G4, >40 years old).

Figure 3

The identification of DEPPs between age groups. Volcano plots showing the most significant differentially expressed proteins (FDR < 0.1 and |log2FC| = 1.5) between G1 and G4 (A), G2 and G3 (B), and G3 and G4 (C). (D) GO enrichment analysis of DEPPs between groups. The top five significant (p-value < 0.05) biological processes, molecular functions, cellular components, and pathways in each group were represented. Age groups: G1, ≤30 years old; G2, 31–35 years old; G3, 36–40 years old; G4, >40 years old.

Figure 4

Protein–protein interaction network between the 25 DEPs (square nodes) and the 60 DEPPs (round nodes) identified in G4 and their regulatory kinases (purple diamond nodes). The green and red outlines represent DEPs upregulated (n = 13) and downregulated (n = 12), respectively. Concerning the DEPPS, the green, red, and dark blue outlines stand for DEPPs with phosphorylated sites upregulated (n = 9), downregulated (n = 47), and both up- and downregulated (n = 4) in G4, respectively. The yellow, blue, and pink round nodes represent DEPs and DEPPs in G4 compared with G1, G2, and G3, respectively. The round node size represents the number of phosphorylated sites altered. All proteins are represented by gene names. Age groups: G1, ≤30 years old; G2, 31–35 years old; G3, 36–40 years old; G4, >40 years old.

Figure 5

Scatterplots displaying the relationship between age and the levels of (A) LYZL1, (B) LAMP1, (C) HSF1, (D) HSP90, (E) HSP27, and (F) p-HSP27/HSP27. Ponceau S was used as the protein-loading control (n = 48).