AB145. Raman spectroscopy as an ex vivo non-invasive approach to distinguish complete and incomplete spermatogenesis within human spermatogenesis

OBJECTIVE: To evaluate the potential clinical application of Raman spectroscopy (RS) as a tool that may identify spermatogenesis within human seminiferous tubules. DESIGN: Raman spectroscopic scanning of human testicular tissue at different maturational stages; immunohistochemistry study and metabolomic analysis of non-obstructive azoospermic/obstructive azoospermic testes. MATERIALS AND METHODS: A total of 52 patients with clinical indications of non-obstructive azoospermia (NOA) and obstructive azoospermia (OA) that underwent infertility evaluation and treatment were collected. This study was approved by the Reproductive Ethics Committee of Renji Hospital, Shanghai Jiao Tong University School of Medicine. Fresh testicular specimens were collected immediately after excision in the operating room and brought to the laboratory of Raman spectroscopy. For Raman scanning, a single seminiferous tubule was placed at the center of a glass bottomed dish and hydrated with PBS solution. Linear equidistant points (a total of 10 points with the interval of 5 µm) at the midline were scanned for each tubule with a 10 seconds period per point. Each testicular sample had ten randomly selected tubules scanned. After completion of Raman scanning, the testicular tissue was immediately fixed in Bouin’s solution and stored in 70% ethanol, embedded in paraffin, sectioned at 5 µm, and stained by hematoxylin and eosin (H&E). The 52 testicular tissue were categoried into three groups: spermatogenesis, maturational arrest (MA), and sertoli cell only syndrome (SCO). Meanwhile, the thickness of lamina propria (LP) was also measured. Then, testicular tissue were studied by immunohistochemistry of type-I, III, IV collagens and laminin. And the differences of metabolites between NOA and OA testes were examined by gas chromatography-mass spectrometer (GC-MS). For data analysis, the real-time Raman spectra were acquired automatically by OPUS 6.5 software and compared among different groups, then the spectral data were then loaded in the Matlab platform for k-means cluster analysis, which aimed to classify the spectra and predict the histological diagnosis for each spectrum. RESULTS: Tubules of OA patients had spectral intensities below 2,000 (au) while tubules of NOA patients had higher intensities, depending on the degree of spermatogenesis. Raman spectroscopy was able to separate samples of NOA and OA testicular tissue with a sensitivity of 90% and specificity of 85.71%. The LP of NOA tubules were thickened, and had increased deposition of type-I and type-III collagens. Twelve detected metabolites showed significant differences between NOA and OA testes. CONCLUSIONS: Raman spectroscopy can non-invasively distinguish seminiferous tubules with complete and incomplete spermatogenesis, and may serve as a novel and potentially useful tool to guide surgeons performing micro-TESE to improve sperm retrieval. SUPPORT: Science and Technology Commission of Shanghai Municipality (No: 10JCI409900), National Basic Research Program of China (No: 2011CB944504) and National Natural Science Foundation of China (Key Program: 31230048).