Integrative Molecular and Functional Analysis of Human Sperm Subpopulations to Identify New Biomarkers of Fertilization Potential

Background: Human ejaculates are composed of sperm subsets with heterogeneous characteristics. Comparative studies of sperm subpopulations with differences in motility may serve to investigate the functional and molecular features that are crucial for reaching the oocyte and fertilizing. Objective: To identify functional and molecular markers that characterize sperm subpopulations with high and low motility. Materials and Methods: Semen samples from 11 donors with proven fertility were processed by density gradient centrifugation to isolate high (F1) and low (F2) motility sperm subpopulations. Besides motility, we evaluated viability, chromatin integrity, mitochondrial membrane potential, capacitation, and acrosomal status. F1 and F2 were subjected to comparative methylome and transcriptome analyses by whole-genome bisulfite sequencing and RNA sequencing, respectively. Further validation of candidate biomarkers at the RNA and protein levels was performed with semen samples from six normozoospermic volunteers using quantitative PCR and Western blotting. Results: Spermatozoa from the F1 fractions exhibited higher mitochondrial membrane potential and viability than F2. Comparative methylome and transcriptome analyses of F1 and F2 identified 271 differentially methylated genes and 82 differentially expressed genes. Notably, CEP128 and CSTPP1 were downregulated and differentially methylated in the F2 fraction. Quantitative PCR confirmed the downregulation of these two genes in F2, and the downregulation of CEP128 was further validated at the protein level by Western blotting. Conclusion: F1 spermatozoa are characterized by elevated mitochondrial membrane potential, viability, and higher expression of CEP128 and CSTPP1. Future studies should evaluate the potential of these functional variables and genes as biomarkers of fertility, either individually or in combination.