1 Departments of Andrology and 2 Radiology, Cairo University Hospitals, Cairo and 3 Adam International Clinic, Giza, Egypt
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Abstract |
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Key words: microdissection/microsurgery/non-obstructive azoospermia/sperm retrieval/testicular biopsy
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Introduction |
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Materials and methods |
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A complete history and physical examinations were taken to evaluate potentially correctable causes of infertility. Hormonal evaluation included serum follicle stimulating hormone (FSH); scrotal ultrasonography was performed before surgery to evaluate testicular size and texture; the testicular blood supply was evaluated by power colour Doppler flow analysis of the testicular parenchyma. Sonographic data were obtained using an Esaote AU3 computed sonography machine with a 7.5 MHz phased array linear probe (Genoa/Florence, Italy).
Surgical approach
Testicular biopsy was carried out under general anaesthesia. Through a small vertical incision in the median scrotal raphe (2 cm), the skin, the dartos muscle and tunica vaginalis were opened to expose the tunica albuginia. The subtunical vessels were identified under the surgical microscope and avoided. A stay suture of 5/0 Prolene was placed into the tunica albuginia, after which a linear transverse (1 cm) incision was made, with care being taken to avoid subtunical vessel injury. The testicular tissues were observed under optical magnification (x24). If no morphologically normal tubules were observed, the incision was extended and blunt dissection performed between the septa of the testicular parenchyma to expose multiple areas. Copious irrigation of the field with Ringer's lactate solution was carried out to prevent blood from obscuring the field, and a small sample was taken from the most dilated tubules, weighed and examined for the presence of spermatozoa. Another two small samples were taken from the adjacent tissues regardless of the tubular diameter; one of these was examined fresh for the presence of spermatozoa, and the other was placed in Bouin's solution for histopathological evaluation. Bipolar diathermy was applied carefully to ensure proper haemostasis. The tunica albugenia was closed using 6/0 Prolene, and the tunica vaginalis was closed with 3/0 chromic catgut. The contralateral testis was exposed via the same incision and a small incision (0.5 cm) was made in the apparently least vascular area to expose the testicular tissues. Gentle pressure was applied to the testis to extrude a sufficient amount of tissue which was excised with sharp scissors, weighed and examined for the presence of testicular spermatozoa. A second piece of tissue was taken for histopathological evaluation. The tunica albuginia was closed using 3/0 chromic catgut. The tunica vaginalis, dartos muscle and skin were closed using 3/0 chromic catgut in layers.
Sperm retrieval
The testicular tissues were taken directly into a Petri dish (Falcon, Cat. no. 3004; Becton Dickinson, Lincoln Park, NJ, USA) containing 1 ml HEPES-buffered Earle's medium. The testicular biopsy was minced and shredded using sterile glass slides and then examined immediately under an inverted microscope (Hoffman optics) using x400 magnification for the presence and motility of testicular spermatozoa. The entire Petri dish was checked. If no spermatozoa were seen, the squeezed tissues were removed from the Petri dish and the remaining medium containing the different tissue cells was collected into 5 ml Falcon tubes. The tubes were centrifuged for 5 min at 300 g. The testicular pellet was resuspended in 2 ml of erythrocyte lysing buffer (Nagy et al., 1997) for 10 min at room temperature, after which 10 ml of HEPES-buffered Earle's medium was added to the specimen in the same tube and centrifuged for 10 min at 500 g. The pellet was resuspended in 50 µl of HEPES-buffered Earle's medium, after which a second search for spermatozoa was performed.
Follow-up
Ultrasonographic examinations were performed at 1, 3 and 6 months after attempts at TESE. Diffuse heterogeneity of the testicular parenchyma or focal hypoechoic lesions were considered acute. Calcifications or linear hyperechoic foci with no associated hypoechogenicity were considered chronic. Testicular blood supply was evaluated using colour Doppler flow analysis of the testicular parenchyma to detect any intratesticular devascularization. Ultrasound examinations were all performed by the same person (R.H.).
Statistical analysis
Statistical analysis was performed using an IBM computer and MS windows statistical program. Descriptive statistics were presented as mean ± SD, as well as frequencies and percentages. Analytical tests used included Student's t-test (two-sided) for comparing the two study groups, and 2 test for contingency table analysis. A P-value of < 0.05 was judged to be statistically significant.
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Results |
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The yield of microsurgical TESE was found to be significantly higher in patients with mixed pathology, which included cases of incomplete spermatogenic arrest, incomplete Sertoli cell-only syndrome and focal tubular sclerosis [20/30 (66.6%) versus 3/30 (10%) (P < 0.05)]. In contrast, there was an insignificant difference between the two approaches in patients with Sertoli cell-only syndrome, primary spermatocyte arrest, early spermatid arrest, tubular hyalinization, hypospermatogenesis and Klinefelter's syndrome.
Sixty patients were followed-up for up to 6 months. Ultrasonography examinations revealed hypoechoic focal testicular lesions one month after the procedures (Table IV). After 6 months, permanent echogenic foci were found less frequently in the microsurgical side than in the conventional side (P < 0.05). Although colour flow Doppler imaging of the testicular parenchyma revealed segmental devascularization, no permanent testicular devascularization could be detected in any patient after 6 months (Table IV
). Testicular volumes remained unchanged during the follow-up period in both sides.
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Discussion |
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In another study (Ostad et al., 1998) that included 81 men with confirmed NOA, sequential biopsy attempts in a therapeutic session were carried out using optical magnifications of x6 to x8 as an approach to minimize injury during TESE. The average number of biopsy attempts was 8.9 for all patients and 6.4 for those in whom spermatozoa were isolated. These authors reported that no evidence of scrotal haematoma or haematocele formation was found postoperatively. Neither was testicular devascularization detected following their approach (Ostad et al., 1998
). In the present study, we did not observe any scrotal haematoma or haematocele in most of the patients who had intraparenchymal haematoma during the follow-up period. Thus, the absence of clinically evident scrotal haematoma does not necessarily guarantee that testicular injury is not present.
Complications following our conventional TESE technique taking only diagnostic samples were lower than in other studies where multiple samples were taken. In an earlier study (Schlegel and Su, 1997), 64 patients were evaluated after TESE for NOA. These authors observed ultrasonographic abnormalities in the testes, suggesting resolving inflammation or haematoma at biopsy sites in 82% (14/18) of patients by one month, and parenchymal calcifications or linear hyperechoic scars in 64.2% (9/14) of patients by 6 months. Two patients had documented impaired testicular blood flow, while one patient had complete devascularization of the testis after TESE with multiple biopsies. In our research, colour flow Doppler imaging of the testicular parenchyma revealed segmental devascularization in 7/60 testes operated on by the conventional approach, and in two testes operated on by the microdissection technique (not significant); however, no permanent testicular devascularization was detected in any testis after 6 months. This finding may be explained by the fact that patients evaluated in the former study (Schlegel and Su, 1997
) were subjected to repeated TESE attempts, while our patients underwent one TESE attempt at which two testicular samples were taken. A previous study (Ron et al., 1998
) had reported focal testicular haematoma in 20/26 (77%) one month after TESE taking up to three samples from different testicular areas. After 6 months, residual focal lesions were detected in 14/26 (54%) of the patients. These authors claimed that the residual focal lesions would be unlikely to have any adverse effect on testicular function (Ron et al., 1998
). However, it is clear that when testicular injury is minimalas in our studythe complications are minimal or absent. In the ICSI era, and with the need for repeated TESE attempts, there is no doubt that such therapeutic procedures would ultimately affect testicular function.
Some authors have advocated the use of biopty gun needle biopsy for extracting testicular spermatozoa from azoospermic patients. The needle can reach a wider area of testicular tissue, and so avoid the possibility that the biopsy sample is taken from a small area that is locally devoid of spermatogenesis (Rajfer and Binder, 1989; Hovatta et al., 1995
; Tuuri et al., 1999
). This technique appears simpler and cheaper, and can be carried out under local anaesthesia compared with our longer procedure which requires general anaesthesia. It was considered as the optimal method for the retrieval of testicular spermatozoa (Tuuri et al., 1999
) giving sufficient amount of tissues and spermatozoa for ICSI, cryopreservation and histopathological evaluation; however, colour Doppler analysis was not performed to verify the safety of this blind procedure against the open technique. Although the microsurgical TESE could be carried out under local anaesthesia at lower cost, shorter recovery period and greater patient satisfaction (Ezeh et al., 1999
), we preferred to use a general anaesthesia in order to avoid any possible vascular complications attributable to local spermatic cord block (Goldstein et al., 1983
; Craft and Tsirigotis, 1995
).
In conclusion, sperm retrieval is increased significantly under optical magnification, although this is not uniform in all testicular histologies. The microdissection TESE is a relatively safe procedure, but is not absolutely devoid of complications. For this reason we prefer to start with a relatively smaller tunical incision, together with meticulous identification of the dilated tubules, before extending the incision to a maximum.
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Acknowledgments |
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Notes |
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References |
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Submitted on May 24, 1999; accepted on November 15, 1999.