Testicular tissue bank: ten years of testicular tissue cryopreservation in Croatia

Abstract

The Testicular Tissue Bank has been operating at the University Hospital Center (UHC) Zagreb since 2013. It aims to cryopreserve testicular tissue from patients with azoospermia. If spermatozoa are found in the collected tissue, a combined procedure known as testicular sperm extraction (TESE)/intracytoplasmic sperm injection (ICSI) is performed. During the last 10 years, our bank has deposited samples from 443 patients collected either by conventional TESE or microsurgical TESE procedure. Among them, 9% were from oncological patients whose samples were stored to preserve fertility. According to pathohistological analysis, 17% of patients were diagnosed with complete spermatogenesis or hypospermatogenesis, 11% with spermatogenic arrest, 48% with mixed atrophy of seminiferous tubules, and 24% with Sertoli cell-only phenotype or tubular fibrosis. Overall, the presence of testicular spermatozoa was found in 58% of patients, which makes them suitable for the ICSI procedure. In 21 out of 59 patients (36%) who underwent a salvage TESE, the outcome was different than that of the first spermatozoa retrieval attempt. Considering that the male factor is one of the leading causes of infertility, the results of Testicular Tissue Bank activity confirm its important role in improving the demographic picture of Croatia.

Figure 1

Patterns of spermatogenesis in azoospermic patients. (A) Full spermatogenesis. Seminiferous tubules are lined with the epithelium, showing all six spermatogenesis stages. The accumulations of late spermatids and spermatozoa are indicated by arrows. Within the loose connective tissue of the testis interstitium are clusters of Leydig cells (L) and some small blood vessels (Bv). Hemalaun & eosin stain (HE), ×200. (B) Hypospermatogenesis. All spermatogenic cells are visible within the seminiferous epithelium, including late spermatids and spermatozoa. However, these cells are not organized in typical spermatogenesis stages (see Figure 1A), and the epithelium stratification is irregular. Despite this, the spermatozoa retrieval rate in these patients, is rather successful (L – Leydig cells; Bv – small blood vessels). HE × 200. (C) Spermatogenic arrest (maturation “stop”). Spermatogenesis stops during the development of a certain spermatogenic cell type. In this case, the development stopped at round spermatids (black arrow). Moreover, many areas of seminiferous tubules demonstrate arrest at the level of primary spermatocytes (white arrow) (Bv – small blood vessels). HE × 200. (D) Sertoli cells-only phenotype (SCO), previously known as Sertoli cells-only syndrome. In this case, the seminiferous epithelium consists exclusively of Sertoli cells. Since this disruption of spermatogenesis has multiple genetic causes (which result in the same phenotype), a new nomenclature has been introduced instead of Sertoli cells-only syndrome. Due to spermatogenic cell loss, there are huge vacuoles (arrow) in Sertoli cells cytoplasm (L – Leydig cells; Bv – small blood vessels). HE × 200. (E) Tubular fibrosis. Seminiferous tubules are devoid of seminiferous epithelium and transformed into connective tissue strands. Between completely fibrosed tubules are abundant clusters of Leydig cells (L) (Bv – blood vessels). HE × 200. (F) Mixed atrophy of seminiferous tubules. This spermatogenesis disorder comprises a combination of seminiferous tubules that display various histological patterns of spermatogenesis (see above). In this case, a tubule with hypospermatogenesis (where all types of spermatogenic cells are present) is seen together with a tubule that is lined only by Sertoli cells [SCO]) (arrow indicates late spermatids and spermatozoa; L – Leydig cells; Bv – small blood vessels). HE × 200.

Figure 2

A flowchart of patients included in the Testicular Tissue Bank. Our bank stored the testicular tissue of 443 patients: 404 underwent the conventional testicular sperm extraction (cTESE), 39 underwent microsurgical TESE (mTESE), 59 underwent salvage treatment (in 40 of them, the previous sperm extraction procedure was performed in an external institution, and in 19 it was performed in the University Hospital Center (UHC) Zagreb. The salvage treatment was performed with cTESE in 46 patients and with mTESE in 13 patients.

Figure 3

The outcome of the salvage procedure compared with the previous testicular sperm retrieval attempt. A successful sperm retrieval attempt is indicated by a red or black bar, depending on whether the spermatozoa were found in the first or salvage procedure. In 21 out of 59 patients who underwent a salvage TESE procedure, the result of the salvage procedure did not match the result of the first testicular sperm retrieval attempt. In 8 patients, spermatozoa were found only during the salvage procedure (circled).