Altered mitochondrial function in sperm from patients with repetitive fertilization failure after ICSI revealed by proteomics.

Torra-Massana, Marc; Jodar, Meritxell; Barragán, Montserrat; Soler-Ventura, Ada; Delgado-Dueñas, David; Rodríguez, Amelia; Oliva, Rafael; Vassena, Rita

BACKGROUND Unexplained fertilization failure (FF), occurring in 1-3% of intracytoplasmic sperm injection (ICSI) cycles, results in both psychological and financial burden for the patients. However, the molecular causes behind FF remain largely unknown. Mass spectrometry is a powerful technique to identify and quantify proteins across samples; however, no study so far has used it to dissect the proteomic signature of sperm with FF after ICSI. OBJECTIVE To investigate whether sperm samples from patients suffering repetitive FF after ICSI display alterations in their protein content. MATERIAL AND METHODS Seventeen infertile men were included: 5 patients presented FF in ≥3 consecutive ICSI cycles, while 12 patients had a fertilization rate >75% (controls). Individual sperm samples were subjected to 2D-LC-MS/MS. Both conventional and novel statistical approaches were used to identify differentially abundant proteins. Additionally, analysis of mitochondrial and proteasomal abundance and activity were performed, using western blot, FACS analysis of JC-1 staining and AMC-peptide fluorometric assay. RESULTS Four proteins presented lower abundance (FMR1NB, FAM209B, RAB2B, PSMA1) in the FF group compared to controls, while five mitochondrial proteins presented higher abundance in FF (DLAT, ATP5H, SLC25A3, SLC25A6, FH) (p<0.05). The altered abundance of mitochondrial DLAT and proteasomal PSMA1 was corroborated by Western-Blot. Of relevance, novel stable-protein pair analysis identified 73 correlations comprising 28 proteins within controls, while different mitochondrial proteins (i.e. PDHA2, PHB2, ATP5F1D) lost >50% of these correlations in specific FF samples pointing out specific mitochondrial deregulations. DISCUSSION This is the first proteomic analysis of sperm from patients who resulted in fertilization failure after ICSI. The altered proteins, most of them related to mitochondrial function, could help to identify diagnostic/prognostic markers of fertilization failure and could further dissect the molecular paternal contribution to reach successful fertilization. CONCLUSION Sperm samples from patients with FF after ICSI present altered abundance of different proteins, including mainly mitochondrial proteins.