Abstract
Varicocele, a condition associated with increased oxidative stress, negatively affects sperm DNA integrity and reduces pregnancy rates. However, the molecular mechanisms related to DNA integrity, damage, and repair in varicocele patients remain unclear. This study aimed to determine the role of DNA repair molecular mechanisms in varicocele-related infertility by combining an in silico proteomics approach with wet-laboratory techniques. Proteomics results previously generated from varicocele patients (n=50) and fertile controls (n=10) attending our Andrology Center were reanalyzed using bioinformatics tools, including the WEB-based Gene SeT AnaLysis Toolkit, Open Target Platform, and Ingenuity Pathway Analysis (IPA), to identify differentially expressed proteins (DEPs) involved in DNA repair. Subsequently, selected DEPs in spermatozoa were validated using western blotting in varicocele (n = 13) and fertile control (n = 5) samples. We identified 99 DEPs mainly involved in male reproductive system disease (n=66) and male infertility (n=47). IPA analysis identified five proteins [fatty acid synthase (FASN), myeloperoxidase (MPO), mitochondrial aconitate hydratase (ACO2), nucleoporin 93 (NUP93), and 26S proteasome non-ATPase regulatory subunit 14 (PSMD14)] associated with DNA repair deficiency, which showed altered expression in varicocele (P <0.03). We validated ACO2 downregulation (fold change=0.37, change%=-62.7%, P=0.0001) and FASN overexpression (fold change = 4.04, change %= 303.7%, P = 0.014) in men with varicocele compared to controls. This study combined a unique in silico approach with an in vitro validation of the molecular mechanisms that may be responsible for varicocele-associated infertility. We identified ACO2 and FASN as possible proteins involved in DNA repair, whose altered expression may contribute to DNA damage in varicocele pathophysiology.
Highlights
Varicocele pathology is characterized by enlargement of the pampiniform plexus, a complex of small spermatic veins that drain venous blood from the testes into the inferior vena cava or the left renal vein
By re-analyzing our previously published proteomics data [26,27,28,29,30,31,32], we identified proteins involved in DNA repair mechanisms in varicocele sperm samples
This is the first study using an in silico approach that identifies proteins involved in DNA repair mechanisms in patients diagnosed with varicocele
Summary
Varicocele pathology is characterized by enlargement of the pampiniform plexus, a complex of small spermatic veins that drain venous blood from the testes into the inferior vena cava (right testicle) or the left renal vein (left testicle). Increased oxidative stress is considered one of the main contributors to infertility in patients with varicocele [3]. This is characterized by high levels of reactive oxygen species (ROS) that can negatively affect sperm functions through the oxidation of lipids, proteins, and DNA, resulting in increased membrane instability, loss of protein function, DNA damage, and reduced pregnancy rate [5, 6]. Considering the impact of oxidative stress on DNA integrity [7], it is not surprising that several studies reported high levels of sperm DNA damage in varicocele patients, regardless of their fertility status [8]. Recent guideline has recommended SDF testing in case of clinical varicocele to help the clinician choosing the best therapeutic plan [8]
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