Cryptorchidism, or undescended testis (UDT), a common congenital anomaly, frequently causes non-obstructive azoospermia (NOA) by impairing spermatogenesis and adult fertility. Microdissection testicular sperm extraction (mTESE) retrieves viable sperm for assisted reproduction, yet reliable predictors of success are lacking, and the molecular mechanisms sustaining active spermatogenesis in UDT remain poorly understood. We analyzed the testicular interstitial fluid (TIF) proteome to identify protein signatures driving spermatogenesis and mTESE outcomes in cryptorchidism, aiming to elucidate the underlying molecular basis of spermatogenic resilience.

Since 2019, TIF was prospectively collected from azoospermic men undergoing mTESE. Eight UDT cases—four with successful sperm retrieval (UDT+) and four without (UDT–)—were compared to six obstructive azoospermia (OA) cases with intact spermatogenesis. TIF underwent liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify differentially expressed proteins. We analyzed UDT versus OA (8 vs. 6) to probe spermatogenic defects and UDT+ versus UDT– (4 vs. 4) to identify proteins linked to successful sperm retrieval.

Of 3172 proteins identified, 490 were shared between UDT and OA, with significant enrichment (p<0.05) of dysregulated stress response and metabolic adaptation proteins in UDT, reflecting adaptive mechanisms to testicular malposition. In UDT, 83 proteins were common to UDT+ and UDT–; UDT+ showed upregulation of cytoskeletal and stress response proteins (e.g., actin-related proteins, HSP70) supporting meiosis and spermatogenesis, while UDT– exhibited enriched proteins tied to impaired microtubule dynamics and stress management (e.g., tubulin isoforms).

TIF proteomic analysis uncovers unique protein expression patterns in cryptorchidism, identifying upregulated cytoskeletal and stress-response proteins as candidate biomarkers for mTESE success. These findings illuminate molecular drivers of spermatogenic resilience in UDT patients with successful sperm retrieval and reveal mechanisms of spermatogenic failure, such as impaired microtubule dynamics, in unsuccessful cases. This work lays a foundation for targeted therapeutic strategies to enhance fertility outcomes.