engleski

Optimalisation of the medium for the cryopreservation of the testicular biopsy

OPTIMALISATION OF THE MEDIUM FOR THE CRYOPRESERVATION OF THE TESTICULAR BIOPSY D. Je`ek, S. Kalanj-Bognar*, @. Vukeli}; ; ; ; *, Lj. Banek, I. Krhen+ and R. Pezerovi}; ; ; ; -Panijan Institute of Histology and Embryology, *Institute of Chemistry and Biochemistry, Medical School University of Zagreb, [alata 3, Zagreb +Urology Clinic, University Clinical Centre "Rebro", Ki{; ; ; ; pati}; ; ; ; eva 11, Zagreb Aim of the study. The cryopreservation of the testicular biopsy is an important step during the treatment of male infertility and assisted reproduction. The well- preserved testicular tissue enables a successful testicular sperm extraction (TESE) and an intracytoplasmic sperm injection into the oocite (ICSI). Since the cryopreservation of the testicular tissue is a rather novel technique, the data on the influence of the cryo-medium on the morphological features of the testicular biopsy are lacking. Our survey was undertaken in order to establish the influence of various cryo- preservation media on the morphology of the testicular biopsy after freezing. Material & methods. Adult male Fischer rats (aged 3 months) were subjected to mild ether anaesthesia. Immediately after the testis isolation, small portions of the testicular tissue were transferred into the transport media (Dulbecco's modified Eagle medium - DMEM and rat serum - RS, 1:1) for 2 hours. Afterwards, the tissue was immersioned into various types of cryomedia (10-50% glycerol/DMEM ; 10% glycerol/10-40% RS/DMEM ; 6-10% glycerol/15- 50% RS/6-10% dimethilsulfoxide - DMSO) and frozen for 24 hours at -75 *C. Finally, the rat testis tissue was frozen in liquid nitrogen (-196 *C) for 2 weeks. After the cryopreservation, the tissue was brought to room temperature (23 *C) and immediately fixed in 4% glutaraldehyde (in 0.1 M phosphate buffer) followed by postfixation in 1% OsO4. The tissue portions were dehydrated and embedded in Durcopan. Semi- and ultrathin sections were made by Reichert ultramicrotome. Ultrathin sections were examined by Carl Zeiss 902A electron microscope (Centre for Electron Microscopy, Medical School University of Zagreb). Results. In general, all testicular samples largely preserved their normal morphology (regardless to the composition of the respective cryo-medium). Only in few seminiferous tubules (where 50% glycerol/DMEM was applied), the spermatogenic cells were delineated from the lamina propria by a "gap". Sertoli cells in such tubules contained small or large translucent vacuoles in their cytoplasm. Spermatogonia and spermatocytes were also divided by small "gaps". However, late spermatids and sperms had normal ultrastructure with normal appearance of the head and the tail. Conclusion. Applied cryo-media (with the exception of 50% glycerol/DMEM) significantly maintained the normal morphology of the cryo-preserved testicular tissue and could be, therefore, used for TESE/ICSI procedure. Literature: 1. Yemini M, Vanderzwalmen P, Mukaida T et al. (1995) Intracytoplasmic sperm injection, fertilisation, and embryo transfer after retrival of spermatozoa by testicular biopsy from an azoospermic male with testicular tubular atrophy. Fertil Steril 63: 1118-1120. 2. Salzbrunn A, Benson DM, Holstein AF et al. (1996) A new concept for the extraction of testicular spermatozoa as a tool for assisted fertilization (ICSI). Hum Reprod 9: 680-683. 3. Je`ek D, Knuth UA, Schulze W. (1998) Successful testicular sperm extraction (TESE) in spite of high serum follicle stimulating hormone and azoospermia: correlation between testicular morphology, TESE results, semen analysis and serum hormone values in 103 infertile men. Hum Reprod 13:1230-1234. Fig. 1. a) Sample of the rat testis cryopreserved in 10% glycerol/DMEM. Sperm tails are visible. x4.500. Bar=1 ľm. b) Sperm tails demonstrate well-preserved ultrastructure. x75.000. Bar=0.5 ľm.

cryopreservation ; testicular biopsy

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