Selection of Individual Testicular Tubules From Biopsied Testicular Tissue With a Stereomicroscope Improves Sperm Retrieval Rate1
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《男科医学杂志》
The Egyptian IVF-ET Center, Cairo, Egypt.
Abstract
The present study describes a new modification for testicular sperm extraction (TESE) with an intraoperative surgical loop, coupled with stereomicroscopic dissection in the laboratory, to identify sperm-containing tubules. The study included 116 consecutive patients with nonobstructive azoospermia (NOA) undergoing TESE and intracytoplasmic sperm injection. After dissection of testicular tissue under the stereomicroscope, patients were separated into 2 groups according to tubular diameter. In the first group (n = 72), all tubules were of the same diameter. In the second group (n = 44), tubules with variable diameters could be identified. In such cases, 1-2 of the most distended and opaque seminiferous tubules were selected and minced alone, then examined for the presence of spermatozoa. The rest of the testicular tissue suspension was minced and examined separately. In 11 (25%) cases, retrieved spermatozoa were found in the isolated distended tubules only. In 21 (47.7%) cases, spermatozoa were found in both the isolated distended tubules and the rest of the specimen. In 2 cases, spermatozoa were found only in the whole sample, not in the isolated tubules. In the remaining 10 cases, spermatozoa were not found in either the whole sample or the isolated tubules. The sperm recovery rate in the isolated tubules was significantly higher than that of the rest of the specimen (72.7% vs 52%, 2 = 3.93, P < .05), and larger numbers of spermatozoa could be easily retrieved in a shorter period of time. In conclusion, the selection and isolation of the most dilated and opaque seminiferous tubules by using the surgical loop, coupled with laboratory stereoscopic dissection, improves sperm retrieval for men with NOA. It is possible that surgical-loops TESE coupled with stereomicroscope may offer superior sperm retrieval when compared with conventional TESE and may also offer reduced operative time when compared with microdissection TESE.
Key words: intracytoplasmic sperm injection, testicular sperm extraction, microdissection, spermatozoa, nonobstructive azoospermia
The advent of intracytoplasmic sperm injection (ICSI) and testicular sperm extraction (TESE) has revolutionized the treatment options to men with nonobstructive azoospermia (NOA). However, success to find spermatozoa in TESE may occur in up to 50%-64% of attempts (Devroey et al, 1995; Kahraman et al, 1996; Mansour et al, 1996, Friedler et al, 1997; Schlegel et al, 1997; Rosenlund et al, 1998). This means that in more than about one third of cases, ICSI will be cancelled due to failure to obtain spermatozoa. In addition the alternative use of spermatid for oocyte injection is rather disappointing (Amer et al, 1997; Vanderzwalmen et al, 1997). In Egypt, as in many other countries, donor sperm is not accepted because of ethical and religious concerns. Therefore, the ICSI cycle is cancelled if no sperm is found. This is why it is of utmost importance to improve the methods and techniques to detect and retrieve testicular spermatozoa in patients with NOA.
Even though TESE is an effective method of sperm retrieval from men with NOA, it has shown many drawbacks. The main drawbacks are the inconsistency of retrieving specimens containing spermatozoa and the residual damage to the testes after the procedure (Schlegel and Su, 1997). This is because the different procedures of TESE are blind approaches without direct vision of the testicular tubules and miniature vasculature.
In order to locate the small foci of sperm-producing tissue in these patients, some authors have described color Doppler ultrasonography in an attempt to determine the most vascular areas, which are hoped to be the most spermatogenicly active areas (Foresta et al, 1998). Other authors have used fine-needle aspiration to map the testes of prospective candidates (Friedler et al, 1997).
In a recently described technique, the use of an operative microscope coupled with a microdissection technique may yield a relatively high sperm retrieval rate with minimal tissue excision (Schlegel, 1999; Amer et al, 2000; Silber, 2000). The procedure for direct microscopic identification of the functioning testicular tubules is referred to as microdissection TESE.
A new modification of this technique with a surgical loop to provide magnification of the testicular tubules during the conventional testicular biopsy, coupled with stereomicroscopic microdissection in the laboratory, is presented here. The aim is to identify sperm-containing tubules and to decrease the cost and operative time.
Materials and Methods
Study Population
One hundred sixteen consecutive patients with NOA undergoing ICSI and TESE were prospectively included in this study. All patients were counseled, and all signed a consent approved by our internal ethical committee. Surgical retrieval of sperm was performed in 88 patients with azoospermia and in 28 patients with severe oligozoospermia, because no normal viable sperm was found in frozen or fresh ejaculates on the day of ova collection.
The routine andrological investigations included conventional semen analysis, endocrine profile, urine analysis, and prostatic examination. Other investigations, including transrectal and scrotal ultrasonography and diagnostic testicular biopsy, were done whenever indicated. Karyotyping, for both partners, was also done to screen for chromosomal abnormalities.
Testicular Biopsy
Testicular biopsy was done under local infiltration anesthesia and cord block with a mixture of 1:1 bupovacaine and lidocaine. In anxious patients, sedation was achieved by injecting medazolam 5 mg IM 15 minutes before surgery. General anesthesia was performed when requested by the patient or when traction on the testes during initial examination caused an intolerable pain, a finding that was common among patients with history of orchiopexy.
Under optical magnification (4x) with a surgical loop, a 3-cm-long median raphe incision was performed. The tunica vaginalis was incised to expose the testis. If massive adhesions were found, or if the epididymis was anteriorly positioned, the wound was extended and the testis was delivered out of the wound. A 5-10 mm incision in the tunica albuginia was performed avoiding vascular areas. The protruding testicular tissue was examined.
If all tubules were of similar diameter and dilated, a biopsy was taken with small sharp scissors, washed with HEPES-buffered Earle's medium to remove blood contaminating the specimen, and transferred immediately to a small Petri dish containing 1-2 mL HEPES-buffered Earle's medium. The excised tissues were dispersed and crushed several times with 2 G30 injection needles. A drop of the suspension was examined immediately in the operating theater. A rapid search for spermatozoa was done, and if no spermatozoa suitable for injection were found, another biopsy was taken either from the same site (1 large biopsy) or from other sites (multiple smaller biopsies), depending on testicular size and the presence of adhesions. A maximum of 3 sites (upper, middle, and lower pole) were taken in order not to compromise subtunical end arteries. The biopsied tissue was rapidly dissected in the operating room and examined once more. If still no spermatozoa were found, another biopsy was performed from the other testis.
In case all the seminiferous tubules were not dilated, the wound was extended to expose a wider area of testicular tissue. Smaller biopsies, 2-3 mm in diameter, were taken from the areas showing dilated tubules. If no dilated tubules were identified, multiple random biopsies were taken from different regions.
If the patient had no previous diagnostic biopsy, or if the slides of his previous diagnostic biopsy were not available for revision in our center, an extra piece of testicular tissue was obtained, fixed in Bouin's solution, and used later to prepare 4-μm-thin paraffin sections stained with hematoxylin and eosin.
Stereomicroscopic Dissection
In the laboratory, each biopsy sample was placed in a Petri dish containing Earle's HEPES-buffered medium and dissected under the stereomicroscope (40x) with G30 needles. One or 2 of the most distended and opaque seminiferous tubules (Figure) were selected and transferred to a separate microdroplet and minced and squeezed with G30 needles, then examined in the injection ICSI dish under the inverted microscope. The rest of the testicular tissue suspension was further minced and squeezed as a whole. Microdroplets from the suspension of the rest of testicular tissue were examined separately for the presence of sperm.
Picture of dilated (A) tubule and collapsed (B) tubule as seen under the stereomicroscope (40x).
Patient Follow-up
Patients were asked to return after 1 week and also after 6 months prospectively for follow-up.
Results
Using the surgical loop, we were able to intraoperatively identify 36 patients (31%) with variable tubular diameter, 53 patients in whom all the testicular tubules were collapsed, and 33 patients in whom all the testicular tubules were dilated. In the laboratory, and by a selection technique with the stereomicroscope, we were able to confirm that the 30 patients with variable diameter did, in fact, have variable diameters of the testicular tubules. In addition, of the 33 specimens in whom all testicular tubules were considered dilated, 6 specimens were considered falsely labeled as dilated tubules and were not opaque. Of the 53 specimens in whom all testicular tubules were collapsed, 14 cases showed dilated tubules under the stereoscope.
Furthermore, we attempted to find a relationship between the testicular histopathology and the size of observed testicular tubules. We found that in the cases in which all testicular tubules were dilated, the diagnosis was usually primary spermatocytes arrest. If all tubules were collapsed, the diagnosis was most likely Sertoli cell only (Table).
Frequency distribution of the main histopathologic patterns during stereoscopic microscopic dissection
In 72 cases, all tubules had the same diameter, whether dilated or collapsed. The sperm recovery in biopsies where all tubules were dilated was 14 of 33 (42%), whereas the sperm recovery was 12 of 53 (23%) when all the tubules were collapsed. In 44 (38% of patients) samples, tubules of variable diameters were found. In 11 cases, spermatozoa were found in the isolated distended tubules only. In 21 cases, spermatozoa were found in both the isolated tubules and the rest of the specimen. In 2 cases, spermatozoa were found only in the rest of the sample but not in the isolated tubules. In 10 cases, no spermatozoa were found in either the rest of the sample or the isolated tubules.
The sperm recovery rate (SRR) was 34 of 44 (72.3%) from isolated distended tubules compared with 52% when whole specimen was examined. Moreover, larger numbers of spermatozoa could be easily retrieved in a shorter period of time from the isolated tubules compared with the whole specimen. We usually find sperm in the isolated distended tubules in less than 5 minutes.
The maximum search time to find spermatozoa in the isolated distended tubule suitable of injecting all available oocytes was 20 minutes. On the other hand, up to 3 hours of searching for enough sperm was needed to examine the rest of the specimen. Regarding the operative time by using our technique, it was 20 ± 12.1 minutes (mean ± SD) in cases of unilateral biopsy (29 cases) and 43.29 ± 10.25 minutes in cases of bilateral biopsy (87 cases). No complications were reported after the procedure, apart from mild to moderate postoperative discomfort and bruises or minor skin infection. No hematocele, orchitis, or gross atrophy was reported.
Discussion
The idea presented in this paper is a compromise between a true microsurgical (or microdissection) TESE and a simple biopsy technique by simply using surgical loops. Our aim in using the surgical loops was to try to identify grossly dilated testicular tubules and to perform a better closure of the incision by trying to avoid the subtunical vessels as much as possible. Our aim was to transfer the fine searching for dilated tubules from the operative field to the laboratory. We selected 1 or 2 of the most dilated testicular tubules and performed mincing separately to save as much time as possible in search for spermatozoa.
In performing TESE for ICSI, andrologists aim to retrieve the largest number of spermatozoa possible with the least tissue excision possible. For this reason, we must adopt newer, more efficient, and less traumatic TESE procedures. Schlegel's (1999) original concept of direct microsurgical inspection of the testicular tubules (microdissection TESE) aimed at minimizing the risk of inadvertent vascular injury to the testis through maximizing the identification of the subtunical vessels, as well as improving the sperm retrieval from men with NOA with minimal tissue excision.
Microdissection TESE was founded on the theory that testicular tubules that contain Sertoli cells alone or fibrotic tubules are thinner and less opaque than tubules that contain spermatozoa in addition to the Sertoli cells (Schlegel, 1999). Success rates have been proved higher with this procedure than with conventional open biopsy TESE and with much less tissue excision (Schlegel, 1999; Okubo et al, 2002). Even the pathological consequences are fewer with TESE by microselection compared with open, classical, surgical biopsies (Amer et al, 2000).
Unfortunately, operative microdissection TESE has several prerequisites that hamper the overall effect. Although the procedure can be done under local anesthesia, many surgeons may prefer general anesthesia. The need for an operative microscope and an experienced andrologist and surgical team with experience in microsurgical techniques may place a dramatic burden on the infertility center. The relatively long operative time and the possible need for general anesthesia increases both the risk of infection and other complications and the cost of the procedure.
In the present study, we used surgical loops instead of the usual operative microscope to detect the presence of dilated testicular tubules that might contain spermatozoa during a conventional biopsy. The procedure was usually done under local anesthesia, and the operative time was plus or minus 20-40 minutes. Under the loop magnification 4x, it was possible to easily identify the different-sized tubules. In addition, intraoperative identification of avascular regions of the testis minimizes the risks of testicular injury. It does carry the usual risks of open conventional multiple biopsy, but this is minimized by the use of surgical loops. Smaller volumes of testicular tissue can be removed with a higher chance of recovering spermatozoa when compared with the standard biopsy procedure.
Thereafter, in the laboratory, a stereomicroscope was used for fine dissection of tubules. Few dilated tubules can be separated and examined separately. The SRR in the isolated tubules was significantly higher (72.7% vs 52%, 2 = 3.93), and larger numbers of spermatozoa could be easily retrieved in a shorter period of time as compared with the conventional TESE, therefore making the TESE procedure easier and improving the overall results.
Schlegel (1999) showed that differential identification of enlarged tubules within the testicular parenchyma was possible in 56% of attempted microdissection TESE procedures. Application of the microdissection technique resulted in an improvement in sperm retrieval rates from 45% per TESE attempt with only standard multibiopsy techniques to 63% with standard biopsy and microdissection attempted in sequential series of TESE procedures (P < .05). For the series of patients who underwent a controlled comparison of standard large biopsies and microdissection, spermatozoa were found only by microdissection but not in standard biopsies in 35% of men where sperm was found.
Furthermore, Amer et al (1999) showed that the total SRR was 56%. Despite removal of a significantly smaller sample of testicular tissues (4.65 ± 3.27 mg vs 53.57 ± 27.45 mg; P < .05), SRR was significantly higher in the side operated under optical magnification (47%) compared with the conventional side (30%; P < .05).
In our series, we were able to identify difference in tubular diameter in 44 of 116 cases (38%). The SSR in the isolated tubules was significantly higher (72.7% vs 52%, 2 = 3.93, P < .05), and larger numbers of spermatozoa could be easily retrieved in a shorter period of time. These results match with the previous reports, even though we used only minimum magnification (4x) with the surgical loop.
Using cryopreservation of testicular spermatozoa before ICSI could be successfully performed to minimize the psychological impact of the procedures and the unnecessary ovulation induction and oocyte retrieval, so long as adequate numbers of spermatozoa are available. However, cryopreservation in cases with severe testicular pathology, where very few sperm are retrieved from a few minute foci, is difficult and may result in total loss of viability. In such cases, spermatozoa may not be retrieved from a repeated TESE. In addition, pregnancy rates have been reported to be lower with cryopreserved sperm from NOA cases than from obstructive cases (Fukunaga et al, 2001).
Several studies reported the physiological consequences of TESE procedure (Shlegel and Su, 1997; Amer et al, 1999, 2000) with testicular ultrasound and color Doppler examination in order to detect intraparenchymal hematoma or segmental devascularization. Most of our patients did not return regularly for follow-up, and because the presence or absence of postoperative discomfort or bruises is not sufficient to support the safety of any procedure, further studies are needed to evaluate if using the surgical loops carries less risk than does conventional TESE. We expect that surgical loops will be superior to conventional TESE but inferior to microdissection TESE.
In conclusion, the selection and isolation of the most dilated and opaque seminiferous tubules by using the stereomicroscope may improve sperm retrieval for men with NOA. This technique does not require any new expensive acquisitions by the IVF center or formal training by the andrologist, and it provides less operative time than does microdissection TESE.
Footnotes
1 A part of this manuscript has been presented orally during the European Society of Human Reproduction and Embryology's 2002 Annual Meeting in Vienna.
References
Amer M, Ateyah A, Hany R, Zohdy W. Prospective comparative study between microsurgical and conventional testicular sperm extraction in non-obstructive azoospermia: follow-up by serial ultrasound examinations. Hum Reprod. 2000; 15:653-656.
Amer M, Haggar SE, Moustafa T, Abd El-Naser T, Zohdy W. Testicular sperm extraction: impact of testicular histology on outcome, number of biopsies to be performed and optimal time for repetition. Hum Reprod. 1999;14:3030-3034.
Amer M, Soliman E, el-Sadek M, Mendoza C, Tesarik J. Is complete spermiogenesis failure a good indication for spermatid conception? Lancet. 1997;350:116.
Devroey P, Liu J, Nagy Z, Goossens A, Tournaye H, Camus M, Van Steirteghem A, Silber S. Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermia. Hum Reprod. 1995; 10:1457-1460.
Foresta C, Garolla A, Bettella A, Ferlin A, Rossato M, Candiani F. Doppler ultrasound of the testis in azoospermic subjects as a parameter of testicular function. Hum Reprod. 1998; 13:3090-3093.
Friedler S, Raziel A, Strassburger D, Soffer Y, Komarovsky D, Ron-El R. Testicular sperm retrieval by percutaneous fine needle sperm aspiration compared with testicular sperm extraction by open biopsy in men with non-obstructive azoospermia. Hum Reprod. 1997; 12:1488-1493.
Fukunaga N, Haigo K, Kyono K, Araki Y. Efficiency of using frozen-thawed testicular sperm for multiple intracytoplasmic sperm injections. J Assist Reprod Genet. 2001; 18:634-637.
Kahraman S, Ozgur S, Alatas C, et al. Fertility with testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermic men. Hum Reprod. 1996; 11:756-760.
Mansour RT, Aboulghar MA, Serour GI, Fahmi I, Ramzy AM, Amin Y. Intracytoplasmic sperm injection using microsurgically retrieved epididymal and testicular sperm. Fertil Steril. 1996; 65:566-572.
Okubo K, Ogura K, Ichioka K, et al. Testicular sperm extraction for nonobstructive azoospermia: results with conventional and microsurgical techniques. Hinyokika Kiyo. 2002; 48:275-280.
Rosenlund B, Kvist U, Ploen L, Rozell BL, Sjoblom P, Hillensjo T. A comparison between open and percutaneous needle biopsies in men with azoospermia. Hum Reprod. 1998; 13:1266-1271.
Schlegel PN. Testicular sperm extraction: microdissection improves sperm yield with minimal tissue excision. Hum Reprod. 1999; 14:131-135.
Schlegel PN, Palermo GD, Goldstein M, Menendez S, Zaninovic N, Veeck LL, Rosenwaks Z. Testicular sperm extraction with intracytoplasmic sperm injection for nonobstructive azoospermia. Urology. 1997; 49:435-440.
Schlegel PN, Su LM. Physiological consequences of testicular sperm extraction. Hum Reprod. 1997; 12:1688-1692.
Silber SJ. Microsurgical TESE and the distribution of spermatogenesis in non-obstructive azoospermia. Hum Reprod. 2000;15:2278-2284.
Vanderzwalmen P, Zech H, Birkenfeld A, et al. Intracytoplasmic injection of spermatids retrieved from testicular tissue: influence of testicular pathology, type of selected spermatids and oocyte activation. Hum Reprod. 1997; 12:1203-1213.(AHMED KAMAL, IBRAHIM FAHM)
Abstract
The present study describes a new modification for testicular sperm extraction (TESE) with an intraoperative surgical loop, coupled with stereomicroscopic dissection in the laboratory, to identify sperm-containing tubules. The study included 116 consecutive patients with nonobstructive azoospermia (NOA) undergoing TESE and intracytoplasmic sperm injection. After dissection of testicular tissue under the stereomicroscope, patients were separated into 2 groups according to tubular diameter. In the first group (n = 72), all tubules were of the same diameter. In the second group (n = 44), tubules with variable diameters could be identified. In such cases, 1-2 of the most distended and opaque seminiferous tubules were selected and minced alone, then examined for the presence of spermatozoa. The rest of the testicular tissue suspension was minced and examined separately. In 11 (25%) cases, retrieved spermatozoa were found in the isolated distended tubules only. In 21 (47.7%) cases, spermatozoa were found in both the isolated distended tubules and the rest of the specimen. In 2 cases, spermatozoa were found only in the whole sample, not in the isolated tubules. In the remaining 10 cases, spermatozoa were not found in either the whole sample or the isolated tubules. The sperm recovery rate in the isolated tubules was significantly higher than that of the rest of the specimen (72.7% vs 52%, 2 = 3.93, P < .05), and larger numbers of spermatozoa could be easily retrieved in a shorter period of time. In conclusion, the selection and isolation of the most dilated and opaque seminiferous tubules by using the surgical loop, coupled with laboratory stereoscopic dissection, improves sperm retrieval for men with NOA. It is possible that surgical-loops TESE coupled with stereomicroscope may offer superior sperm retrieval when compared with conventional TESE and may also offer reduced operative time when compared with microdissection TESE.
Key words: intracytoplasmic sperm injection, testicular sperm extraction, microdissection, spermatozoa, nonobstructive azoospermia
The advent of intracytoplasmic sperm injection (ICSI) and testicular sperm extraction (TESE) has revolutionized the treatment options to men with nonobstructive azoospermia (NOA). However, success to find spermatozoa in TESE may occur in up to 50%-64% of attempts (Devroey et al, 1995; Kahraman et al, 1996; Mansour et al, 1996, Friedler et al, 1997; Schlegel et al, 1997; Rosenlund et al, 1998). This means that in more than about one third of cases, ICSI will be cancelled due to failure to obtain spermatozoa. In addition the alternative use of spermatid for oocyte injection is rather disappointing (Amer et al, 1997; Vanderzwalmen et al, 1997). In Egypt, as in many other countries, donor sperm is not accepted because of ethical and religious concerns. Therefore, the ICSI cycle is cancelled if no sperm is found. This is why it is of utmost importance to improve the methods and techniques to detect and retrieve testicular spermatozoa in patients with NOA.
Even though TESE is an effective method of sperm retrieval from men with NOA, it has shown many drawbacks. The main drawbacks are the inconsistency of retrieving specimens containing spermatozoa and the residual damage to the testes after the procedure (Schlegel and Su, 1997). This is because the different procedures of TESE are blind approaches without direct vision of the testicular tubules and miniature vasculature.
In order to locate the small foci of sperm-producing tissue in these patients, some authors have described color Doppler ultrasonography in an attempt to determine the most vascular areas, which are hoped to be the most spermatogenicly active areas (Foresta et al, 1998). Other authors have used fine-needle aspiration to map the testes of prospective candidates (Friedler et al, 1997).
In a recently described technique, the use of an operative microscope coupled with a microdissection technique may yield a relatively high sperm retrieval rate with minimal tissue excision (Schlegel, 1999; Amer et al, 2000; Silber, 2000). The procedure for direct microscopic identification of the functioning testicular tubules is referred to as microdissection TESE.
A new modification of this technique with a surgical loop to provide magnification of the testicular tubules during the conventional testicular biopsy, coupled with stereomicroscopic microdissection in the laboratory, is presented here. The aim is to identify sperm-containing tubules and to decrease the cost and operative time.
Materials and Methods
Study Population
One hundred sixteen consecutive patients with NOA undergoing ICSI and TESE were prospectively included in this study. All patients were counseled, and all signed a consent approved by our internal ethical committee. Surgical retrieval of sperm was performed in 88 patients with azoospermia and in 28 patients with severe oligozoospermia, because no normal viable sperm was found in frozen or fresh ejaculates on the day of ova collection.
The routine andrological investigations included conventional semen analysis, endocrine profile, urine analysis, and prostatic examination. Other investigations, including transrectal and scrotal ultrasonography and diagnostic testicular biopsy, were done whenever indicated. Karyotyping, for both partners, was also done to screen for chromosomal abnormalities.
Testicular Biopsy
Testicular biopsy was done under local infiltration anesthesia and cord block with a mixture of 1:1 bupovacaine and lidocaine. In anxious patients, sedation was achieved by injecting medazolam 5 mg IM 15 minutes before surgery. General anesthesia was performed when requested by the patient or when traction on the testes during initial examination caused an intolerable pain, a finding that was common among patients with history of orchiopexy.
Under optical magnification (4x) with a surgical loop, a 3-cm-long median raphe incision was performed. The tunica vaginalis was incised to expose the testis. If massive adhesions were found, or if the epididymis was anteriorly positioned, the wound was extended and the testis was delivered out of the wound. A 5-10 mm incision in the tunica albuginia was performed avoiding vascular areas. The protruding testicular tissue was examined.
If all tubules were of similar diameter and dilated, a biopsy was taken with small sharp scissors, washed with HEPES-buffered Earle's medium to remove blood contaminating the specimen, and transferred immediately to a small Petri dish containing 1-2 mL HEPES-buffered Earle's medium. The excised tissues were dispersed and crushed several times with 2 G30 injection needles. A drop of the suspension was examined immediately in the operating theater. A rapid search for spermatozoa was done, and if no spermatozoa suitable for injection were found, another biopsy was taken either from the same site (1 large biopsy) or from other sites (multiple smaller biopsies), depending on testicular size and the presence of adhesions. A maximum of 3 sites (upper, middle, and lower pole) were taken in order not to compromise subtunical end arteries. The biopsied tissue was rapidly dissected in the operating room and examined once more. If still no spermatozoa were found, another biopsy was performed from the other testis.
In case all the seminiferous tubules were not dilated, the wound was extended to expose a wider area of testicular tissue. Smaller biopsies, 2-3 mm in diameter, were taken from the areas showing dilated tubules. If no dilated tubules were identified, multiple random biopsies were taken from different regions.
If the patient had no previous diagnostic biopsy, or if the slides of his previous diagnostic biopsy were not available for revision in our center, an extra piece of testicular tissue was obtained, fixed in Bouin's solution, and used later to prepare 4-μm-thin paraffin sections stained with hematoxylin and eosin.
Stereomicroscopic Dissection
In the laboratory, each biopsy sample was placed in a Petri dish containing Earle's HEPES-buffered medium and dissected under the stereomicroscope (40x) with G30 needles. One or 2 of the most distended and opaque seminiferous tubules (Figure) were selected and transferred to a separate microdroplet and minced and squeezed with G30 needles, then examined in the injection ICSI dish under the inverted microscope. The rest of the testicular tissue suspension was further minced and squeezed as a whole. Microdroplets from the suspension of the rest of testicular tissue were examined separately for the presence of sperm.
Picture of dilated (A) tubule and collapsed (B) tubule as seen under the stereomicroscope (40x).
Patient Follow-up
Patients were asked to return after 1 week and also after 6 months prospectively for follow-up.
Results
Using the surgical loop, we were able to intraoperatively identify 36 patients (31%) with variable tubular diameter, 53 patients in whom all the testicular tubules were collapsed, and 33 patients in whom all the testicular tubules were dilated. In the laboratory, and by a selection technique with the stereomicroscope, we were able to confirm that the 30 patients with variable diameter did, in fact, have variable diameters of the testicular tubules. In addition, of the 33 specimens in whom all testicular tubules were considered dilated, 6 specimens were considered falsely labeled as dilated tubules and were not opaque. Of the 53 specimens in whom all testicular tubules were collapsed, 14 cases showed dilated tubules under the stereoscope.
Furthermore, we attempted to find a relationship between the testicular histopathology and the size of observed testicular tubules. We found that in the cases in which all testicular tubules were dilated, the diagnosis was usually primary spermatocytes arrest. If all tubules were collapsed, the diagnosis was most likely Sertoli cell only (Table).
Frequency distribution of the main histopathologic patterns during stereoscopic microscopic dissection
In 72 cases, all tubules had the same diameter, whether dilated or collapsed. The sperm recovery in biopsies where all tubules were dilated was 14 of 33 (42%), whereas the sperm recovery was 12 of 53 (23%) when all the tubules were collapsed. In 44 (38% of patients) samples, tubules of variable diameters were found. In 11 cases, spermatozoa were found in the isolated distended tubules only. In 21 cases, spermatozoa were found in both the isolated tubules and the rest of the specimen. In 2 cases, spermatozoa were found only in the rest of the sample but not in the isolated tubules. In 10 cases, no spermatozoa were found in either the rest of the sample or the isolated tubules.
The sperm recovery rate (SRR) was 34 of 44 (72.3%) from isolated distended tubules compared with 52% when whole specimen was examined. Moreover, larger numbers of spermatozoa could be easily retrieved in a shorter period of time from the isolated tubules compared with the whole specimen. We usually find sperm in the isolated distended tubules in less than 5 minutes.
The maximum search time to find spermatozoa in the isolated distended tubule suitable of injecting all available oocytes was 20 minutes. On the other hand, up to 3 hours of searching for enough sperm was needed to examine the rest of the specimen. Regarding the operative time by using our technique, it was 20 ± 12.1 minutes (mean ± SD) in cases of unilateral biopsy (29 cases) and 43.29 ± 10.25 minutes in cases of bilateral biopsy (87 cases). No complications were reported after the procedure, apart from mild to moderate postoperative discomfort and bruises or minor skin infection. No hematocele, orchitis, or gross atrophy was reported.
Discussion
The idea presented in this paper is a compromise between a true microsurgical (or microdissection) TESE and a simple biopsy technique by simply using surgical loops. Our aim in using the surgical loops was to try to identify grossly dilated testicular tubules and to perform a better closure of the incision by trying to avoid the subtunical vessels as much as possible. Our aim was to transfer the fine searching for dilated tubules from the operative field to the laboratory. We selected 1 or 2 of the most dilated testicular tubules and performed mincing separately to save as much time as possible in search for spermatozoa.
In performing TESE for ICSI, andrologists aim to retrieve the largest number of spermatozoa possible with the least tissue excision possible. For this reason, we must adopt newer, more efficient, and less traumatic TESE procedures. Schlegel's (1999) original concept of direct microsurgical inspection of the testicular tubules (microdissection TESE) aimed at minimizing the risk of inadvertent vascular injury to the testis through maximizing the identification of the subtunical vessels, as well as improving the sperm retrieval from men with NOA with minimal tissue excision.
Microdissection TESE was founded on the theory that testicular tubules that contain Sertoli cells alone or fibrotic tubules are thinner and less opaque than tubules that contain spermatozoa in addition to the Sertoli cells (Schlegel, 1999). Success rates have been proved higher with this procedure than with conventional open biopsy TESE and with much less tissue excision (Schlegel, 1999; Okubo et al, 2002). Even the pathological consequences are fewer with TESE by microselection compared with open, classical, surgical biopsies (Amer et al, 2000).
Unfortunately, operative microdissection TESE has several prerequisites that hamper the overall effect. Although the procedure can be done under local anesthesia, many surgeons may prefer general anesthesia. The need for an operative microscope and an experienced andrologist and surgical team with experience in microsurgical techniques may place a dramatic burden on the infertility center. The relatively long operative time and the possible need for general anesthesia increases both the risk of infection and other complications and the cost of the procedure.
In the present study, we used surgical loops instead of the usual operative microscope to detect the presence of dilated testicular tubules that might contain spermatozoa during a conventional biopsy. The procedure was usually done under local anesthesia, and the operative time was plus or minus 20-40 minutes. Under the loop magnification 4x, it was possible to easily identify the different-sized tubules. In addition, intraoperative identification of avascular regions of the testis minimizes the risks of testicular injury. It does carry the usual risks of open conventional multiple biopsy, but this is minimized by the use of surgical loops. Smaller volumes of testicular tissue can be removed with a higher chance of recovering spermatozoa when compared with the standard biopsy procedure.
Thereafter, in the laboratory, a stereomicroscope was used for fine dissection of tubules. Few dilated tubules can be separated and examined separately. The SRR in the isolated tubules was significantly higher (72.7% vs 52%, 2 = 3.93), and larger numbers of spermatozoa could be easily retrieved in a shorter period of time as compared with the conventional TESE, therefore making the TESE procedure easier and improving the overall results.
Schlegel (1999) showed that differential identification of enlarged tubules within the testicular parenchyma was possible in 56% of attempted microdissection TESE procedures. Application of the microdissection technique resulted in an improvement in sperm retrieval rates from 45% per TESE attempt with only standard multibiopsy techniques to 63% with standard biopsy and microdissection attempted in sequential series of TESE procedures (P < .05). For the series of patients who underwent a controlled comparison of standard large biopsies and microdissection, spermatozoa were found only by microdissection but not in standard biopsies in 35% of men where sperm was found.
Furthermore, Amer et al (1999) showed that the total SRR was 56%. Despite removal of a significantly smaller sample of testicular tissues (4.65 ± 3.27 mg vs 53.57 ± 27.45 mg; P < .05), SRR was significantly higher in the side operated under optical magnification (47%) compared with the conventional side (30%; P < .05).
In our series, we were able to identify difference in tubular diameter in 44 of 116 cases (38%). The SSR in the isolated tubules was significantly higher (72.7% vs 52%, 2 = 3.93, P < .05), and larger numbers of spermatozoa could be easily retrieved in a shorter period of time. These results match with the previous reports, even though we used only minimum magnification (4x) with the surgical loop.
Using cryopreservation of testicular spermatozoa before ICSI could be successfully performed to minimize the psychological impact of the procedures and the unnecessary ovulation induction and oocyte retrieval, so long as adequate numbers of spermatozoa are available. However, cryopreservation in cases with severe testicular pathology, where very few sperm are retrieved from a few minute foci, is difficult and may result in total loss of viability. In such cases, spermatozoa may not be retrieved from a repeated TESE. In addition, pregnancy rates have been reported to be lower with cryopreserved sperm from NOA cases than from obstructive cases (Fukunaga et al, 2001).
Several studies reported the physiological consequences of TESE procedure (Shlegel and Su, 1997; Amer et al, 1999, 2000) with testicular ultrasound and color Doppler examination in order to detect intraparenchymal hematoma or segmental devascularization. Most of our patients did not return regularly for follow-up, and because the presence or absence of postoperative discomfort or bruises is not sufficient to support the safety of any procedure, further studies are needed to evaluate if using the surgical loops carries less risk than does conventional TESE. We expect that surgical loops will be superior to conventional TESE but inferior to microdissection TESE.
In conclusion, the selection and isolation of the most dilated and opaque seminiferous tubules by using the stereomicroscope may improve sperm retrieval for men with NOA. This technique does not require any new expensive acquisitions by the IVF center or formal training by the andrologist, and it provides less operative time than does microdissection TESE.
Footnotes
1 A part of this manuscript has been presented orally during the European Society of Human Reproduction and Embryology's 2002 Annual Meeting in Vienna.
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