ࡱ> 7 YybjbjUU .7|7|ol(((()I)))))U/U/U/HHHHHHH$^J ~LI]U/.NU/U/U/I@))ObI@@@U/Z ))H@U/H@@3G^3G)) wd_$(<23G3GxI0I3GxM>xM3G@From Testicular Biopsy to Human Embryo D. Je~ek1, N. Kne~evi2, S. Kalanj-Bognar3, }. Vukeli3 and I. Krhen2 1 University of Zagreb, Medical School, Dept. of Histology and Embryology 2 University of Zagreb, Medical School, Clinic for Urology 3 University of Zagreb, Medical School, Dept. of Chemistry and Biochemistry Address for correspondence: As. Prof. Davor Je~ek, M.D., Ph.D. University of Zagreb Medical School Dept. of Histology and Embryology `alata 3, HR-10000 ZAGREB CROATIA, EUROPE Phone/Fax.: +385 1 45 90 251 e-mail: davor@mef.hr Abstract The aim of the study was to investigate the role of a testicular biopsy in the diagnosis and therapy of infertile men with a non-obstructive azoospermia. Overall, 70 testicular biopsies from infertile men were analysed. Samples were obtained by the open testicular biopsy method. After dissection, several pieces of the tissue were immediately immersed into the Sperm Prep Medium (Medi-Cult) and fixative (5.5% buffered glutaraldehyde). Tissue samples transported in Sperm Prep Medium were plunged into Sperm Freezing Medium (Medi-Cult) and were stored in liquid nitrogen for potential in vitro fertilization procedures. The tissue was also processed for semi-thin sections and transmission electron microscopy. Semi-thin sections from 8 infertile patients demonstrated regular testis structure and fully preserved spermatogenesis (control biopsies). In the remaining 62 cases, spermatogenesis was impaired and a variety of pathological changes could be seen: disorganization and desquamation of spermatogenic cells, spermatid or spermatocyte stop, spermatogonia only, Sertoli cells only or tubular fibrosis. However, in 65% of cases (despite the above mentioned changes of seminiferous epithelium) foci of preserved spermatogenesis could be detected. These cases were classified as mixed atrophy of seminiferous tubules. In 63% of infertile patients, a successful extraction of sperm from the biopsy could be performed. In azoospermic patients, histological analysis of testicular biopsy proved to be very useful in terms of diagnosis as well as therapy, i.e. further in vitro fertilization procedures. Introduction It is estimated that infertility affects 15% of all couples trying to conceive. Epidemiological studies have pointed out that half of infertility problems could be related to the male factor. In many cases (30-70%) the cause for male infertility is not known or not clarified enough (19,31). Conventional in vitro fertilization methods (IVF) are not useful for patients with extreme oligospermia or azoospermia. Application of partial zona dissection (3), zona drilling (11), and subzonal insemination (18) instead of conventional IVF offered only limited success in treatment. Therefore, the introduction of intracytoplasmic sperm injection into the oocyte (ICSI) significantly improved the treatment of infertile men with extremely poor number or no sperm into the semen (21). Successful fertilization of human oocytes by intracytoplasmic sperm injection was reported by Lanzendorf and co-workers (17). In 1992, Palermo and collaborators (22) reported delivery of the first ICSI-conceived infant. ICSI has had a significant impact on human IVF, particularly in the treatment of idiopathic infertility. In cases of azoospermic patients with congenital bilateral absence of the vas deferens or acquired obstruction, sperm could be obtained surgically, by microepididymal sperm aspiration (MESA) or percutaneous sperm aspiration (PESA). Temple-Smith and co-workers (37) performed IVF using sperm obtained by MESA and reported the first pregnancy by microepididymal sperm aspiration-IVF. The outcome of such treatment has been rather poor, because epididymal sperm motility is generally low when compared to that of ejaculated sperm. The application of ICSI markedly improved fertilization using epididymal sperm (39). Percutaneous sperm aspiration (PESA) was developed to avoid postoperative problems often caused by MESA. This method is simple to perform and good pregnancy rates using this technique have been achieved (4,5,32). However, percutaneous sperm aspiration may damage the tissue because of the sightless puncturing. That is why the method has been modified by making a small surgical window on the scrotum. This enabled satisfactory sperm yield without much tissue damage (16). If there is no possibility to retrieve sperm from epididymis by MESA and PESA, one has to apply other methods. Testicular sperm aspiration (TESA) is, like PESA, rather simple to perform. An 18-21-gauge needle is punctured into the testis in order to retrieve sperm and could be made on an outpatient basis (2,14). The technique involves blind needle puncture, and if one fails to identify sperm in the isolated tissue, several punctures with a needle are needed. No significant postoperative complications after this approach have been reported (6). However, in male rats it was recently demonstrated that TESA caused irreversible multifocal damage of seminiferous tubules in contrast to the open biopsy of the testis (33). The first fertilization by ICSI using testicular sperm was reported by Craft and collaborators in 1993 (7) and the first pregnancy using testicular sperm was reported by Schoysman and co-workers in 1993 (28). Oocytes were retrieved from six patients with excretory azoospermia, and subzonal insemination and ICSI were performed. One biochemical and one ongoing pregnancy were achieved after embryo transfer in six cycles. It was concluded that testicular sperm has the ability to fertilize, and the embryos fertilized by testicular sperm were capable of establishing viable pregnancies. Another technique that enables to yield sperm from the testis is a testicular sperm extraction (TESE). This method involves open testicular biopsy instead of a needle aspiration. Thus, several pieces of testicular tissue could be obtained, microdissected and used for TESE/ICSI, or stored in liquid nitrogen for future TESE/ICSI attempts (9). In the meantime, histological analysis of the biopsy could be preformed. It should be pointed out that, before ICSI, testicular biopsy was mostly used for diagnostic purposes. Nowadays, it is also used for the therapy of the male infertility. Moreover, freezing of testicular biopsy and storage in liquid nitrogen enables to form a bank of testicular biopsies for each patient/couple and to retrieve such a biopsy when needed (15,29,30). The purpose of this paper is to discuss the role of testicular biopsy in diagnosis and therapy of male infertility and assisted reproduction as well as to present own data on TESE. Materials & methods 70 couples that consulted andrologist at the Urology Clinic (University of Zagreb, Medical School) for infertility in the period 1997-2003 were included in the current study. The infertility problem was due to the male factor. In 62 infertile men, a non-obstructive azoospermia could be diagnosed. 8 patients had obstructive azoospermia with completely preserved testicular tissue and served as controls. All patients were subjected to the open biopsy of the testis (13). Whenever possible, a bilateral biopsy was performed. A detailed diagnostic procedure preceded testicular biopsy. Some diagnostic steps in the diagnosis of azoospermia are enlisted in Table 1. Briefly, an incision of 8-10 mm in length in the t. albuginea of the testis has been made. This incision allows 4-5 testicular lobules to be included into the biopsy. The protruding testicular tissue was dissected using surgical microscissors. Several pieces were taken from different parts of the male gonad. After dissection, all pieces were immediately transferred into a transport medium (Sperm-Prep, Medi-Cult) and brought to the Dept. of Histology and Embryology (University of Zagreb, Medical School) for consequent freezing and histological analysis. Thus, each piece of testicular tissue was divided into two parts: one part was immersed in 0.5 ml Sperm Freezing Medium (Medi-Cult) and another fixed in a buffered 5.5% glutaraldehyde. Tissue in the Sperm Freezing Medium was subjected to a programmed freezing using a Nicool LM 10 freezing device (Air Liquid). During first 5 min. the tissue was cooled up to -70C, then exponentially to -120C in the following 55 min. Finally, the tissue samples were stored in the liquid nitrogen until the TESE/ICSI attempt. Tissue fixed in glutaraldehyde was rinsed several times in 0.05M phosphate buffer (pH=7.1-7.4, 800 mOsm), postfixed with 1%OsO4 and dehydrated. After a routine histological procedure, the testicular tissue was embedded in Durcopan (Agar). Semi-thin sections (section thickness = 0.9 m) were made by Reichert ultramicrotome and stained with 1% toluidine blue. The sections were analysed by Nikon binocular microscope (Eclipse E200). In some cases, ultra-thin sections were made (section thickness = 70 nm), contrasted with lead citrate and uranyl acetate and examined by transmission electron microscope Zeiss 902A (Centre for Electron Microscopy, Medical School Univ. of Zagreb). The histological evaluation of the testicular biopsy was performed using a modified Johnsen score (Table 2) (8). For a planned TESE/ICSI attempt, the testicular sperm extraction has been made as described below. Frozen samples were retrieved form the liquid nitrogen and immediately transferred into a water bath (37C). Sperm Freezing Medium (Medi-Cult) was replaced with the prewarmed (37C) 1 ml Sperm-Prep Medium (Medi-Cult) and incubated for 1 hour. This was followed by adding 1 ml of the prewarmed Sperm - Prep Medium with 0.8 mg collagenase (Type IA, Sigma) and 0.2 g trypsine inhibitor (Sigma). After 2 hours of incubation, samples were macerated with sterile needles, centrifuged for 10 min. at 800g and 37C. After the removal of a supernatant, the pellet was checked for the presence of sperm. Results Histological analysis demonstrated that the control group had normal structure of seminiferous tubules as well as the interstitial compartment of the testis. Seminiferous tubules had diameter ranging from 170 to 210 m. Lamina propria consisted of 5-7 layers of peritubular (myoid) cells. Tubules were lined with seminiferous epithelium, resting on the basement membrane of a normal thickness. Within the seminiferous epithelium, Sertoli cells had indented nucleus with a prominent nucleolar complex. The cytoplasm of these cells contained few lipid droplets and vacuoles. Spermatogenic cells consisted of spermatogonia, primary and secondary spermatocytes, round and elongated spermatids. Occasionally, sperm could be noted released into the lumen of tubules. Leydig cells in the interstitial tissue had oval or round nucleus, sometimes with a prominent nucleolus. Abundant cytoplasm contained few lipid droplets. Within the cytoplasm of some cells, Reinkes crystal/s could be noted. Blood vessels had normal thickness of t. intima, media and externa. Infertile group of patients presented various degrees of testicular damage. In 65% (n= 40) a mixed atrophy of seminiferous tubules has been observed. This testicular damage involves seminiferous tubules with different morphology (34): some tubules had normal morphology with full spermatogenesis going on; in some tubules seminiferous epithelium was lacking and the tubules were transformed into the fibrous tissue (so-called tubular shadows); a number of seminiferous tubules were lined with Sertoli cells or spermatogonia exclusively; finally, some tubules had seminiferous epithelium composed of primary and secondary spermatocytes and, rarely, round and elongated spermatids. In short, there was a mosaic picture of testicular damage. In one case, only a diffuse tubular fibrosis has been found by histology. In 20% of infertile patients (n= 12), Sertoli cells only syndrome could be diagnosed. In these cases, seminiferous epithelium was reduced to supportive Sertoli cells. These cells had often changed morphology: their nucleus had no indentations, became more oval (instead of pear-shaped) and cytoplasm possessed numerous lipid droplets, vacuoles and phagocytozed material. Some of the cells were more stained with toluidine blue, which gave a dark aspect of the cytoplasm and nucleus. In 15% (n= 9) of analysed cases, the seminiferous epithelium was partially maintained and consisted of spermatogonia, spermatocytes, round and elongated spermatids. However, these cells were very often found to be disorganized and desquamated into the lumen of tubules. They rested on the thickened basement membrane and lamina propria. Testicular interstitial compartment in the biopsies of infertile patients displayed a variety of changes. Some Leydig cells had normal appearance with regular, round or oval nucleus and few lipid droplets in their cytoplasm. Occasionally, Reinkes crystals could be found in their cytoplasm. Other Leydig cells in the infertile group had very often indented nucleus, many transparent vacuoles and numerous lipid droplets. No or few Reinkes crystals could be seen in such changed cells. Blood vessels of the interstitium sometimes preserved their morphology: thin t. intima, neatly organized t. media and moderately developed t. externa. However, in a number of cases blood vessels had thickened t. intima and, sometimes, t. media. Within the interstitium of infertile group of patients one could also note increased presence of mast cells and macrophages. After enzymatic digestion with collagenase and maceration of the tissue, TESE was positive in 63% (n=39) (positive isolation of at least 5 sperm). Negative result (no sperm found) was recorded in 37% (n=23) of infertile patients. Discussion The diagnostic approach to an infertile couple should be a comprehensive one, with a detailed diagnostic procedure (24). Testicular biopsy, if necessary, should be at the very end of this procedure. Some major indications for testicular biopsy are given in Table 3. During the operation, it is highly recommended to take biopsy from various sites, to freeze several pieces of the tissue and to perform a histological analysis. There is an on-going discussion whether to perform TESA or TESE procedure (10,23,40). In our opinion, TESA should be performed in the case of obstructive azoospermia, when other methods to acquire sperm (i.e. PESA/MESA) have failed. Even with one or two aspirations with an 18-21 gauge needle, a satisfactory sperm yield could be accomplished. However, in the case of non-obstructive azoospermia, TESE procedure is recommended. The open biopsy of testicular tissue can, in such cases, be used for histological diagnosis and biopsy frozen for further consequent TESE/ICSI attempts (15,25,29,30). Examination of seminiferous tubules under the operating microscope and their microdissection during operation could additionally increase sperm yield by TESE (1,26,27,35,36). The comparison between TESA and TESE is given in Table 4. Another important issue is the accuracy of the histological analysis. Various parts of the testis may have different histological appearance, i.e. preserved or damaged spermatogenesis (12,20). That is why it is crucial to take open biopsy of the testis from at least 3 different sites. Histological analysis of only one biopsy (piece) could be misleading and a wrong diagnosis could be established, since frozen biopsies may have a quite different morphology. In order to increase the accuracy of the histological analysis, a concept of sandwich organisation of the testicular biopsy was proposed (15,30). According to this concept, one piece of testicular tissue is subjected to the histological analysis and another to enzymatic digestion/maceration (so-called diagnostic TESE). Data of both analyses are combined in order to put the correct diagnosis (Fig.1a). However, even when such analysis is done, there could be doubts on the morphology of the frozen parts. Therefore, we would like to propose a new way for the analysis of the testicular biopsy which we called piece per piece. Namely, each piece of dissected testicular tissue is divided into 2 parts: one part is always histologically evaluated and another part frozen. Histological analysis is made on minimum of 50 serial semi-thin sections per block, thus ensuring sufficient tissue sections for histological evaluation. The diagnostic TESE is avoided because a more precise diagnosis could be done on sections. Usually 3 pieces per testis are frozen and subjected to histology (total = 6 pieces). The above-described concept is illustrated on Fig.1b. Enzymatic digestion contributes to the sperm yield form frozen parts of the biopsy. It was shown that mild concentration of collagenase produces defects in the lamina propria of seminiferous tubules, thus releasing sperm trapped in the tubular lumen (25). Additional maceration of the biopsy with sterile needles significantly increases the presence of sperm in the pellet (29,30). If there is no sperm in the sample, elongated or round spermatids could be used for ICSI (38). However, despite some optimistic reports on round spermtid injection into the oocyte, sperm and elongated spermatids are cells of preference. Therefore, the pellet should be carefully observed in order to identify the above-mentioned cells. This may take sometimes 1-2 hours. Future investigations are necessary to establish which sperm isolated from the testicular biopsy is the most suitable one for ICSI. Recent studies on testicular sperm DNA damage rouse significant concern (31). New methods for selection of viable and undamaged sperm (or elongated/round spermatid) for ICSI could be very helpful in order to avoid unnecessary ovarian stimulation and TESE/ICSI attempts. More comprehensive preventive measures to reduce infertility should be taken. These include the reduction of pollution (estrogen-like substances), change in the life style and diminishing of stress in developed countries. Improvement of spermatogenic cell cultivation (spermatogonia, spermatocytes, round spermatids) could be applied when sperm are lacking in the testicular biopsy. Investigations on embryonic stem cells, their cultivation and development into sperm are exciting future perspectives for the treatment of infertility. The same goes for the differentiation of embryonic tumour cells. New genetic and protein markers for identifying infertility should be found employing new techniques like DNA multiarray analysis and proteomics. Acknowledgements The study was supported by Das Partnerschaftsabkommen der Med. Fakultt der Universitt Zagreb und des FB Medizin der Universitt Hamburg. The valuable help of Prof. Dr. W. Schulze and Prof. Dr. H.J. Seitz is gratefully acknowledged. References 1. Amer M, Ateyah A, Hany R, Zohdy W (2000) Prospective comparative study between microsurgical and conventional testicular sperm extraction in non-obstructive azoospermia: follow-up by serial ultrasound examination. Hum Repord 15:653-656 2. Bourne H, Watkins W, Sperirs A, Baker HWG (1995) Pregnacies after intracytoplasmic injection of sperm collected by fine needle biopsy of the testis. Fertil Steril 64:433-435 3. Cohen J, Malter H, Fehilly C, Wright G, Elsner C, Kort H (1998) Implantation of embryos after partial zona opening of oocyte zona pellucida to faciliate sperm penetration. Lancet 2: 162 4. Craft I, Khalifa Y, Tsirigotis M, Hogewind G, Bennett V, Nicholson N (1995) Perctuaneous epididymal sperm aspiration and intracytoplasmic sperm injection in the management of infertility due to obstructive azoospermia. Fertil Steril 63:1038-1042 5. Craft I, Khalifa Y, Boulos A, Pelekanos M, Foster C, Tsirigotis M (1995) Factors influencing the outcome of in-vitro with percutaneous aspirated epididymal spermatozoa and intracytoplasmic injection in azoospermic men. Hum Reprod 10:1791-1794 6. Craft I, Tsirigotis M (1995) Simplified recovery, preparation and cryopreservation of testicular spermatozoa. Hum Repord 10:1623-1626 7. Craft I, Bennett V, Nicholson N (1993) Fertilizing ability of testicular spermatozoa. Lancet 342:864 8. DeKretser DM, Holstein AF (1976) Testicular biopsy and abnormal germ cells. In: Hafez ESE (ed.) Human Semen and Fertility Regulation In Men. Mosby, St. Louis, pp. 332-343 9. Fischer R, Baukloh V, Naether OGJ (1996) Pregnancy after intracytoplasmic sperm injection of spermatozoa extracted from frozen-thawed testicular biopsy. Hum Reprod 11:2197-2199 10. Friedler S, Raziel A, Strassburger D, Soffer Y, Komarowsky D, Ron-El R (1997) Testicular sperm retrieval by percutaneous fine needle aspiration compared with testicular sperm extraction by open biopsy in men with non-obstructive azoospermia. Hum Reprod 12: 1488-1493 11. Gordon JW, Grunfeld J, Garrisi GJ, Talansky BE, Richards C, Laufer N (1988) Fertilization of human oocytes by sperm from infertile males after zona pellucida drilling. Fertil Steril 50: 68-73 12. Hauser R, Botchan A, Amit A, Ben Yosef D, Gamzu R, Paz G, Lessing JB, Yogev L, Yavetz H (1998) Multiple testicular sampling in non-obstructive azoospermias is it necessary? Hum Reprod 13: 3081-3085 13. Holstein AF, Wulfhekel U (1971) Die Semidnnschnitt-Technik als Grundlage freine cytolgische Beurteilung der Spermatogenese des Menschen. Andrologia 3:65-69 14. Hovatta O, Moilanen J, Von Smitten K, Reima I (1995) Testicular needle biopsy, open biopsy, epididymal aspiration and intracytoplasmic sperm injection in obstructive azoospermia. Hum Reprod 10:2595-2599 15. Je~ek D, Knuth UA, Schulze W (1998) Successful 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 fertile men. Hum Reprod 13: 1230-1234 16. Kwang-Yul C, Ki-Boong O, Hyun-Joo K (1997) Approaches for obtaining sperm in patients with male factor infertility. Fertil Steril 67:985-995 17. Lanzendorf SM, Slusser J, Maloney MK, Hodgen GD, Veeck LL, Rosenwaks Z (1988) A preclinical evaluation of pronuclear formation by microinjection of human spermatozoa into human oocytes. Fertil Steril 49:835-842 18. Ng S-C, Bongso TA, Ratnam SS, Sathananthan AH, Chan CLK, Wong PC (1988) Pregnacy after transfer of multiple sperm under the zona. Lancet 2:790 19. Oehninger S (2001) Strategies for the infertile man. Semin Reprod Med 19: 231-237 20. Ostad M, Liotta D, Ye Z, Schlegl PN (1998) Testicular sperm extraction (TESE) for non-obstructive azoospermia: results of multibiopsy approach with optimized tissue dispersion. Urology 52: 692-696 21. Palermo GD, Adler A, Cohen J, Rosenwaks Z, Alikani M (1995) Intracytoplasmic sperm injection: a novel treatment for all forms of male factor infertility. Fertil Steril 63:1231-1240 22. Palermo G, Joris H, Devroey P, Van Steirteghem AC (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340: 17-18 23. Rosenlund B, Kvist U, Plen L, Ekstrom U, Hovatta O (1998) A comparison between open and percutaneous needle biopsies in men with azoospermia. Hum Reprod 13:1266-1271 24. 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Silber SJ, Nagy ZP, Liu J, Godoy H, Devroey P, Van Steirteghem AC (1993) Conventional in vitro fertilization versus intracytoplasmic sperm injection for patients requiring microsurgical sperm aspiration. Hum Reprod 9:1705-1709 36. Silber SJ (2000) Microsurgical TESE and the distribution of spermatogenesis in non-obstructive azoospermia. Hum Reprod 15:2278-2284 37. Temple-Smith PD, Southwick GJ, Yates CA, Trounson AO, De Kretser DM (1995) Human pregnancy by in vitro fertilization (IVF) using sperm aspirated from the epididymis. J In Vitro Fert Embryo Transf 2:119-122 38. Tesarik J, Mendosa C, Testart J (1995) Viable embryos from injection of round spermatid into oocytes. N Engl J Med 333:525 39. Tournaye H, Nagy Z, Devroey P, Lissens W, Liu J, Van Steirteghem AC (1994) Microsurgical epididymal sperm aspiration and intracytoplasmic injection: a new effective approach to infertility as a result of congenital bilateral absence of the vas deferens. Fertil Steril 61:1045-1051 40. Turek P, Cha I, Ljung BM (1997) Systematic fine-needle aspiration of the testis:correlation to biopsy and results of organ mapping for mature sperm in azoospermic men. Urology 49:743-748 Table 1. Some diagnostic steps in the case of male factor infertility. Testicular biopsy should be considered as final step, if appropriate. DIAGNOSTIC STEPAnamnesis & status (testis consistency, volume) Hormones: FSH, LH, prolactin, testosterone, inhibin-B PSA Detailed ejaculate analysis Urine analysis (retrograde ejaculation) Microbiology (ejaculate, urethral smear, hepatitis, AIDS) Karyotype Y chromosome microdeletions Cystic fibrosis (in situ hybridization) Other relevant examinations Testicular biopsy  Table 2. Evaluation of the testicular biopsy using Johnsen modified score. SCOREMORPHOLOGY10Full spermatogenesis9Many late spermatids, disorganized tubular epithelium8Few late spermatids7No late spermatids, many early spermatids6No late spermatids, arrest at spermatid level5Many spermatocytes, no spermatids4Arrest at primary spermatocyte level, no spermatids3Spermatogonia only2Sertoli cells only1Tubular sclerosis Table 3. Indications for testicular biopsy. INDICATIONBilateral aplasia of d. deferens Idiopathic normogonadotrophic azoospermia Hypergonadotrophic azoospermia Ejaculation failure (resistant to treatment) Cryptozoospermia Necrozoospermia Inoperable postesticular obstruction Operation of postesticular obstruction  Table 4. Comparison between TESA and TESE procedure. TESATESESimple, fast, outpatient basis Local anaesthesia Cytological smear used for diagnosis Multiple, often must be repeated Sometimes no result despite positive cytology Sometimes must be simultaneous with oocyte aspirationComplex, more expensive Local/general anaesthesia Method of choice in obstructive, non-obstructive or tumour-induced azoospermia Semi-thin sections/histology mostly used for diagnosis Detection of Ca in situ Only one procedure is sufficient Treatment of women is time-independent from the operation of a man (frozen biopsies are used) TESE/ICSI could be repeated without a new operation due to biopsy freezing  Fig. 1. Analysis of testicular biopsy. A)Sandwich principle; B) Piece to piece principle. 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