4.1. Patient Recruitment

The present study included forty patients diagnosed with azoospermia or cryptozoospermia who underwent testicular biopsy as part of their assisted reproduction treatment at the Embryolab Fertility Clinic (Thessaloniki, Greece). Written informed consent was obtained from all participants, and the study was approved by the Ethics Committee of the University of Thessaly.

Azoospermia was diagnosed after at least two semen analyses conducted according to World Health Organization (WHO) guidelines. A comprehensive diagnostic process was followed, including a medical history assessment, physical examination, and scrotal imaging. All participants also completed a detailed questionnaire about their medical history, lifestyle factors, etc. (File S1). Further investigations included genetic tests for karyotype, DF508 mutation, Y-chromosome microdeletions, and serum hormone measurements (follicle-stimulating hormone, luteinizing hormone, and testosterone). Only patients in whom no identifiable cause of male infertility was found were included in this study.

During testicular sperm extraction (TESE), experienced urologists recovered multiple tissue samples from both testicles, which were mechanically dissected and microscopically examined for the presence of mature spermatozoa. Biopsies were categorized based on the abundance of spermatozoa as follows: (i) “No presence”—no spermatozoa observed after at least 30 min of searching under an inverted microscope by three experienced operators; (ii) “Rare presence”—spermatozoa found after more than 10 min of searching; and (iii) “High presence”—spermatozoa identified within 2 min of searching [115]. Furthermore, in this study, cases of cryptozoospermia, in which TESE was also performed to achieve assisted reproduction, were also included. For small RNA sequencing, a small portion of each testicular tissue sample was collected in sterile Falcon tubes and stored at −80 °C until shipment on dry ice to the Laboratory of Genetics, Comparative and Evolutionary Biology (University of Thessaly, Larissa, Greece), for RNA extraction and further processing of the samples. The patients were followed-up to assess pregnancy status, and the samples were further categorized based on spermatozoa presence in the testes tissue and pregnancy outcomes following assisted reproduction techniques, as follows: Cryptozoospermia (n = 8), no presence of spermatozoa (n = 8), rare presence of spermatozoa—no pregnancy (n = 8), rare presence of spermatozoa—pregnancy (n = 8), and high presence of spermatozoa (n = 8).