Proteomic Analysis of Testicular Interstitial Fluid in Men with Azoospermia

Abstract

Background: The primary microenvironment of the testis comprises testicular interstitial fluid (TIF) surrounding the seminiferous tubules and testicular interstitial tissue. The pathological alterations of germ and Sertoli cells could affect the TIF composition and might contain putative biomarkers for monitoring active spermatogenesis.

Objective: We identified differentially expressed proteins in the TIF of patients with obstructive (OA) or nonobstructive (NOA) azoospermia to elucidate the underlying etiology of defective spermatogenesis.

Design setting and participants: We prospectively enrolled nine patients, including three men with OA and six with NOA with (n = 3) and without (n = 3) successful sperm retrieval. Their TIF was collected during the testicular sperm extraction procedure.

Outcome measurements and statistical analysis: TIF was analyzed using liquid chromatography-tandem mass spectrometry to identify differentially expressed proteins specific to OA and NOA with or without successful sperm retrieval. The dysregulated protein was further validated using Western blotting.

Results and limitations: Among the 555 TIF proteins identified in NOA patients, 14 were downregulated relative to OA patients. These proteins participate in biological processes such as proteolysis, complement activation, and immune responses; complement and coagulation cascade pathways were also enriched. Furthermore, 68 proteins with significantly higher levels were identified in the TIF of NOA patients with successful sperm retrieval than in those with failed sperm retrieval; these are mainly implicated in oxidation-reduction processes. The expression of calreticulin, which can distinguish successful and failed testicular sperm retrieval in the NOA group, was validated by Western blotting.

Conclusions: We provide the first scientific evaluation of TIF protein composition in men with azoospermia. These findings will help identify the physiological and pathological roles of each protein in regulating sperm production. Thus, our study underscores the potential of TIF in sperm retrieval biomarker discovery and would serve as a foundation for further studies to improve treatment strategies against azoospermia.

Patient summary: Using a proteomic approach, we identified and analyzed the total protein content of testicular interstitial fluid in humans with defective spermatogenesis for the first time and discovered altered protein expression patterns in patients with nonobstructive azoospermia (NOA). Proteins related to oxidation-reduction processes were upregulated in NOA patients with successful sperm retrieval compared with those with failed sperm retrieval. This can aid the development of novel diagnostic tools for successful testicular sperm retrieval.

Keywords: Azoospermia; Proteomic; Testicular interstitial fluid.

Fig. 1

Obtaining testicular interstitial fluid (TIF) while performing microdissection testicular sperm extraction. (A) Maintain firm compression on testis using the thumb and index finger from the start of the procedure, prior to incising the tunica albuginea, throughout the entire TIF collection process. (B) Magnified image obtained during the TIF collection procedure, using a 20× magnification to provide a more detailed view of the steps involved.

Fig. 2

Schematic workflow for evaluating the TIF composition in men with azoospermia. LC-MS/MS = liquid chromatography tandem mass spectrometry; TIF = testicular interstitial fluid.

Fig. 3

TIF proteome result of the label-free experiment. (A) Venn diagram comparing TIF proteins found to be enriched ± two-fold in group 2 versus group 1. (B) Enriched GO biological process, cellular component, and molecular function in 14 downregulated and 142 upregulated proteins. GO = gene ontology; NOA = nonobstructive azoospermia; OA = obstructive azoospermia; TIF = testicular interstitial fluid.

Fig. 4

Comparison of TIF proteins identified in groups 2 and 3. (A) Venn diagram comparing TIF proteins found to be enriched ± two-fold in group 3 versus group 2. (B) Enriched GO biological process, cellular component, and molecular function in 16 downregulated and 68 upregulated proteins. GO = gene ontology; NOA = nonobstructive azoospermia; TIF = testicular interstitial fluid.

Fig. 5

C3 and C4 levels in TIF in OA and NOA patients. Group 1: obstructive azoospermia. Group 2: nonobstructive azoospermia with unsuccessful sperm retrieval. NOA = nonobstructive azoospermia; OA = obstructive azoospermia; TIF = testicular interstitial fluid.

Fig. 6

Western blot analysis of calreticulin in the TIF of OA and NOA patients. Group 1: obstructive azoospermia; group 2: nonobstructive azoospermia with successful sperm retrieval; and group 3: nonobstructive azoospermia without successful sperm retrieval. NOA = nonobstructive azoospermia; OA = obstructive azoospermia; TIF = testicular interstitial fluid.