Abstract. Introduction. RBMOnline - Vol 18. No Reproductive BioMedicine Online; on web 8 January 2009

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1 RBMOnline - Vol 18. No Reproductive BioMedicine Online; on web 8 January 2009 Article Microsurgical TESE versus conventional TESE for ICSI in non-obstructive azoospermia: a randomized controlled study Giovanni M Colpi, MD, is a specialist in Urology and Andrology. He is Head of the Andrological-Urology Unit and IVF Centre, San Paolo Hospital, Milan, and Visiting Professor in Andrology of the Universities of Pavia, L Aquila, and Milan. His clinical research focuses on new techniques in andrological surgery, tests in seminology, sonography and tumours in male infertility, ICSI in azoospermia, semen cryopreservation and the perineal floor in erectile dysfunction. He is an active member of a range of international societies and has authored or edited many international and Italian publications. Dr Giovanni M Colpi Giovanni M Colpi 1,4, Elisabetta M Colpi 2, Guido Piediferro 1, Daniela Giacchetta 1, Giacomo Gazzano 1, Fabrizio M Castiglioni 2, M Cristina Magli 3, Luca Gianaroli 3 1 Andrological-Urology Unit and IVF Center, San Paolo Hospital, University of Milano, Italy; 2 Istituto per la Sterilità e la Sessualità, Milano; 3 International Institute of Reproductive Medicine, Lugano, Switzerland 4 Correspondence: gmcolpi@yahoo.com Abstract In a population of non-obstructive azoospermia patients, the efficacy of microsurgical testicular sperm extraction (microtese) and conventional TESE was evaluated in a randomized controlled study on 138 testicles, classified and paired in a 48-square table according to the different classes of the following three variables: patient plasma FSH concentration, orchidometry and testicular histology. Sperm retrieval was positive in 21/22 testicles with hypospermatogenesis (11/11, 10/11; microtese, TESE respectively), in 12/14 with maturation arrest (6/7, 6/7), in 16/22 with incomplete Sertoli cell-only syndrome (8/11, 8/11), and in 16/80 with complete Sertoli cell-only syndrome (11/40, 5/40). Sperm recovery was positive in 5/24 patients with FSH concentration 3 maximum value of normal range (N) (4/12, 1/12), in 17/40 patients with 2N FSH < 3N (9/20, 8/20), in 30/48 patients with N < FSH < 2N (17/24, 13/24), and in 13/26 patients with FSH = N (6/13, 7/13). Regarding orchidometry, sperm recovery was positive in 11/18 testicles with volume (V) 12 ml (6/9, 5/9), in 27/56 testicles with 8 ml V < 12 ml (15/28, 12/28), and in 27/64 testicles with V < 8 ml (15/32, 12/32). FSH value and the surgical procedure were the two variables significantly (P < 0.05) predicting positive sperm retrieval. Keywords: FSH concentration, microtese, non-obstructive azoospermia, orchidometry, TESE, testicular histology Introduction The surgical retrieval of spermatozoa to be used for intracytoplasmic sperm injection (ICSI) cycles remains the only possibility of fathering a child for patients suffering from non-obstructive azoospermia (NOA). A recent review of the scientific literature shows that acceptable recovery rates are obtained with single testicular sperm extraction (TESE) (49.5%; Donoso et al., 2007), multiple TESE (52.5%; Colpi et al., 2005), and microsurgical TESE (microtese) (47 64%; Colpi et al., 2005; El-Haggar et al., 2007; Talas et al., 2007). Conversely, the testicular fine needle aspiration technique is by far less efficacious (10.0%; El-Haggar et al., 2007) and therefore discouraged by the Guidelines of the European Association of Urology (Dohle et al., 2007). There are only a few studies reporting on the outcome of multiple TESE, and this is possibly due to the fact that the slight advantage in the recovery rate associated with this procedure does not compensate for its greater invasiveness. Although not randomized, several studies have been published on the comparison between microtese and TESE, according to which microtese yields a higher recovery rate (Schlegel, 1999; Amer et al., 2000; Okada et al., 2002; Okubo et al., 2002; Tsujimura at al., 2002, 2006). The aim of this study was to verify the efficacy of microtese compared with TESE in retrieving spermatozoa from NOA patients in a controlled randomized study Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK

2 316 Materials and methods Patients and inclusion criteria From June 2004 to December 2006, 154 NOA patients underwent either TESE or microtese. Patients included in the study were allocated, according to the waiting list, to general anaesthesia procedure (i.e. microtese) or to day surgery (i.e. TESE) on the basis of the general operative theatre plan, after informed consent including explanations about results in the literature and invasiveness of the two procedures, for a total of 195 explored testicles. The age of patients ranged between 19 and 57 years (mean years; median 36 years). Inclusion criteria were: (i) azoospermia proven by at least two semen analyses (including microscopical examination of the pellet after centrifugation, followed by further examination of the re-centrifuged upper fluid), performed according to the World Health Organization (1999) criteria in the study centre s laboratory of seminology during the previous 12 months, at an interval of at least 3 months and after at least 2 months of good health; (ii) a normal karyotype; (iii) a normal serum testosterone concentration; and (iv) no Y-chromosome microdeletions in peripheral leukocytes. The week before surgery, all patients were submitted to an accurate evaluation of the volume of each testis (i.e. orchidometry) by ultrasonography (volume [ml] = 0.52 A B C, A, B and C being the measurements [cm] of the three testicular axes as they were detected ultrasonographically). Plasma FSH concentration was also measured. Each patient was first operated on the testis with the better volume. When spermatozoa were not found after an initial search of 10 min (the biologist started to examine the removed testicular tissue directly in the operating room), surgery was started on the second testis. Nevertheless, due to the economic policy of the hospital, the second testis surgery was always performed as TESE. A total of 117 testicles underwent TESE procedures, while microtese was performed on 78 testicles. Surgical procedure MicroTESE was performed through a transversal incision of the testis covering three-quarters of its circumference, according to a line preserving as much as possible the predominantly transversal subalbugineal vessels. The testis was opened like a book by gently separating the lobular tissue of both sides with a spatula. Then, the tissue was observed under the microscope at magnification to search for areas with dilated tubules, from which numerous microretrievals were performed, according to the protocol described by Schlegel (1999). When no diversity in the tubular diameters was observed in the different areas, the microretrievals were performed according to a sort of mapping, by removing tiny fragments of testicular tissue from the two separated surfaces, at different depths from the albuginea to the hilum. The fragments were washed in human tubal fluid medium to remove the blood, and given to the biologist for the microscopic examination. Only afterwards, the testicular tissue surfaces were irrigated for antisepsis with Ringer solution (plus 80 mg gentamycin/100 ml). Haemostasis was then performed by gently pressing the testicular tissue for 2 min using gauze wet with the above antiseptic solution, and eventually using a microsurgical bipolar thermal device. The albuginea incision was closed with a continuous suture of Vicryl 5/0. The TESE was performed by removing a large fragment of testicular tissue of about mm through a transversal incision of the albuginea, either equatorially or in the cranial part of the testis. Antisepsis with Ringer solution containing gentamycin, haemostasis with coagulator, and continuous closure of the albuginea with Vicryl 4/0 were performed as described above. The total quantity of testicular tissue that was removed from each testis by TESE or microtese was approximately the same. In both procedures, the tunica vaginalis opening was repaired by a continuous Vicryl 4/0 after instillation into the vaginalis cavity of 1.5 mg betamethasone (a 2-ml ampoule) to prevent pain and tunica vaginalis adhesions. Biological search for spermatozoa The tiny fragments of testicular tissue were placed in sterile Petri dishes with 0.5 ml sperm washing medium (Irvine Scientific, Santa Ana, CA, USA) and meticulously minced using microscissors. The resulting microfragments were then passed through a Venflon for 3 5 min, until a homogeneous pulverized suspension was obtained, according to Schlegel s protocol (1999). Small aliquots of the suspension were directly examined under a microscope for the presence of spermatozoa. The search could last up to 4 h. In all cases, the biological search was performed by the same operator (DG) and the result was given as positive or negative. When the fluid obtained after the suspension s centrifugation showed more than 100 spermatozoa/mm 3, the viability of the retrieved spermatozoa was assessed by eosin nigrosin live/dead stain test. Testicular histology At every surgical procedure, either TESE or microtese, a fragment of subcapsular parenchyma (about mm) was removed soon after incising the albuginea, then fixed in Bouin s solution and sent to the pathologist. Histological analysis was conducted by examining at least 100 different sections of tubuli seminiferi. The histological results were defined as: (i) complete Sertoli cell-only syndrome (cscos) when the tubules were populated by only Sertoli cells; (ii) incomplete SCOS (iscos) when germ cells were present in a few tubules, while the majority showed only Sertoli cells; (iii) maturation arrest (MA) characterized with an arrest of the spermatogenetic maturation sequence; and (iv) hypospermatogenesis in which tubules showed a severely reduced population of germ cells and a poor order of spermatogenesis. All the histological examinations were performed by the same pathologist (GG). Hormonal dosage During the 2 months preceding the surgical intervention, all patients were tested for FSH, LH, testosterone, and prolactin in qualified laboratories, relating their values to the maximum

3 value of the normal range. In the present study, only the plasma FSH concentrations were considered. Data collection and randomization Every operated testicle was classified according to the following variables: (i) testicular volume (V), categorized according to volume (< 8 ml, 8 ml V < 12 ml, and 12 ml, i.e. normal); (ii) FSH concentration, categorized according to multiples of the normal range (N) ( 3 maximum value of the normal range, 2N FSH < 3N, N < FSH < 2N, and N; (iii) testicular histology, categorized according to definition (cscos, iscos, MA and hypospermatogenesis). All testicles undergoing TESE (117) or microtese (78) were therefore classified according to the above-mentioned variables in a 48-square table. Each testicle of every single square of the TESE group was paired with a testicle of the corresponding square of the microtese group following the criterion of randomization of the alphabetical order of the patients last name. At the end, the biological archives were opened and the results of presence or absence of spermatozoa in the surgical sperm retrieval were added to the squares table. Statistical analysis Data were evaluated by a technique of binary logistic regression with statistical significance set at P < Results Table 1 shows the results of the presence or absence of spermatozoa after the surgical retrievals by microtese and TESE, which were randomly paired for data that were homogeneous for class of testicular volume, class of FSH concentration and class of histology. In 80/138 paired testicles (58%), cscos was diagnosed. This high percentage was probably due to the fact that this study s population included patients referred by other centres, being considered as poor prognosis due to small orchidometry and/or high FSH concentration. Of the remaining testicles, 22 (16%) were affected by iscos, 14 (10%) by MA, and 22 (16%) by hypospermatogenesis. Regarding the orchidometry, of 138 testicles, 56 (40.6%) had a volume between 8 and 12 ml; 18 (13.0%) were greater than 12 ml, and 64 (46.4%) were smaller than 8 ml. Only 26/138 (18.8%) testicles came from patients with normal FSH concentration; 48 (34.8%) belonged to patients with N < FSH < 2N; 40 (29.0%) to patients with 2N FSH < 3N; and 24 (17.4%) to patients with FSH 3N. The retrieval of spermatozoa was positive in 21/22 testicles affected by hypospermatogenesis (95.4%; 11/11 with microtese and 10/11 with TESE); in 12/14 affected by MA (85.7%; 6/7 both with microtese and TESE); in 16/22 (72.7%) affected by iscos (8/11 both with microtese and with TESE); and in 16/80 (20.0%) of those affected by cscos (11/40 with microtese and 5/40 with TESE). Sperm recovery was positive in 5/24 (20.8%) patients with FSH 3N (4/12 with microtese, 1/12 with TESE); in 17/40 (42.5%) with 2N FSH < 3N (9/20 with microtese and 8/20 with TESE); in 30/48 (62.5%) patients with N < FSH < 2N (17/24 with microtese and 13/24 with TESE); and in 13/26 (50.0%) patients with FSH = N (6/13 with microtese and 7/13 with TESE). In all, sperm recovery was positive in 43/74 (58.1%) patients with FSH < 2N. The percentages of recovery in the different classes of FSH concentration by microtese versus TESE are represented in Figure 1. Regarding the testicular volume, sperm recovery was positive in 11/18 testicles (61.1%) with V 12 ml (6/9 with microtese, 5/9 with TESE); in 27/56 testicles (48.2%) with 8 ml V < 12 ml (15/28 with microtese, 12/28 with TESE); and in 27/64 testicles (42.2%) with V < 8 ml (15/32 with microtese, 12/32 with TESE). Figure 2 shows the percentage of recovery in the different classes of testicular volume. According to the statistical analysis, FSH value and the surgical procedure were the two variables that could significantly predict a positive sperm retrieval (P < 0.05). The testis volume and histology were shown to play a less important role. To evaluate the viability of the retrieved spermatozoa, only 36 samples could be submitted to eosin nigrosin test (19 from microtese and 17 from TESE group). The mean sperm viability was 43.5% (median 43%, range 74 21%) in microtese samples and 41.6% (median 42%, range 86 19%) in TESE samples. Discussion The few authors comparing the results obtained by microtese and TESE in NOA patients (Schlegel, 1999; Amer et al., 2000; Okada et al., 2002; Tsujimura at al., 2002) have reported a higher efficacy by microtese in yielding positive sperm recovery, even when multiple TESE is performed (Okada et al., 2002; Tsujimura et al., 2002). Some authors refer an increased efficacy by microtese in cases of MA and SCOS (Okada et al., 2002), associated with a lower frequency (Amer et al., 2000; Okada et al., 2002) or total absence of complications (Dardashti et al., 2000; Okubo et al., 2002; Tsujimura et al., 2002). Unfortunately, none of these clinically excellent reports are controlled randomized studies. Some authors (Schlegel, 1999; Okada et al., 2002; Tsujimura et al., 2002; Ramasamy et al., 2005) have compared the two techniques in groups of NOA patients, who had a nonhomogeneous prognosis due to the different distribution of testicular histological patterns. Other authors used TESE on one testicle and microtese on the other testicle after assuming that the histology was comparable (Amer et al., 2000). In other reports (Okubo et al., 2002), the microtese was performed only when the TESE had given negative sperm recovery, using the microtese as a rescuing technique in case of negative sperm recovery with standard TESE (Tsujimura et al., 2006). According to preliminary experience of NOA patients who underwent TESE and, after extending the incision, microtese, there was a difference in favour of microtese (13/22 versus 9/22) (Colpi et al., 2005). 317

4 Table 1. Presence or absence of spermatozoa after retrieval by microtese or TESE from testicles randomly paired according to class of testicular volume, plasma FSH concentration and histology. FSH Testis volume cscos iscos MA Hypospermatogenesis Total a concentration (V) (ml) 3N <8 5 (1), 5 (0) 1 (1), 1 (1) 12 (3, 2:1) 8 but <12 3 (1), 3 (0) 2 (1), 2 (0) 10 (2, 2:0) 12 1 (0), 1 (0) 2 (0, 0:0) 2N but <3N <8 10 (2), 10 (2) 1 (1), 1 (1) 1 (1), 1 (0) 1 (1), 1 (1) 26 (9, 5:4) 8 but <12 3 (0), 3 (0) 1 (1), 1 (1) 2 (2), 2 (2) 1 (1), 1 (1) 14 (8, 4:4) 12 N but <2N <8 6 (3), 6 (1) 2 (2), 2 (2) 1 (1), 1 (1) 1 (1), 1 (1) 20 (12, 7:5) 8 but <12 5 (2), 5 (1) 1 (1), 1 (1) 3 (3), 3 (2) 18 (10, 6:4) 12 2 (1), 2 (1) 1 (1) 1 (1) 1 (1), 1 (1) 1 (1), 1 (1) 10 (8, 4:4) N <8 1 (0), 1 (0) 1 (0), 1 (1) 1 (1), 1 (1) 6 (3, 1:2) 8 but <12 2 (0), 2 (0) 2 (1), 2 (1) 1 (0), 1 (1) 2 (2), 2 (2) 14 (7, 3:4) 12 2 (1), 2 (0) 1 (1), 1 (1) 6 (3, 2:1) Total a 80 (16; 11:5) 22 (16; 8:8) 14 (12; 6:6) 22 (21; 11:10) 138 (65; 36:29) In each comparison, a testicle that had been operated by microtese was randomly paired to a testicle operated by TESE. Numbers in brackets indicate the number of cases that had a positive recovery of spermatozoa. The data from TESE are in italics. cscos = complete Sertoli cell-only syndrome; iscos = incomplete SCOS; MA = maturation arrest; N = maximum value of normal range. a Values are: total number of testicles (total number of positive cases; microtese:tese). Figure 1. Percentage of positive sperm retrieval according to method of sperm extraction (microtese versus TESE) in relation to FSH concentration. N = maximum value of normal range. Figure 2. Percentage of positive sperm retrieval according to method of sperm extraction (microtese versus TESE) in relation to testicular volume. 318 As far as is known, this is the first controlled study that compares TESE and microtese and relates the positive sperm recovery to three variables, FSH concentration, orchidometry, and testicular histology, which are all clinically relevant for NOA patients. The highest efficacy of microtese could be due to the possibility of: (i) selecting larger tubules (110 µm is the smallest tubule diameter permitting successful sperm retrieval; Amer et al., 2008) and taking biopsies in the more vascularized areas as supported by others (Schlegel, 1999): this strategy not being possible with TESE; and (ii) recovering testicular microfragments in different areas of the testicle in a sort of testicular mapping, while for TESE a large monofocal biopsy is performed. Furthermore, microtese provides the possibility of performing biopsies close to the hilum testis. Although still theoretical, it is believed that spermatogenetic areas could be more frequent in this region. A future controlled study is planned to verify this hypothesis. MicroTESE is more expensive than TESE in terms of surgical time, special sutures and dedicated equipment. Nevertheless, no surgical complications at all resulted in a series of 260 cases from 1999 to November In addition, the follow-up of a large series of the authors cases at 3 and 6 months after the operation demonstrated no visible damage to the testicles, to the point that very frequently the scar was not visible at ultrasound scanning, and no significant alteration of the hormonal functionality of the testicle resulted (Ramasamy et al., 2005).

5 As for NOA patients the surgical retrieval represents the only hope of conceiving, microtese appears to be recommendable especially in cases of high FSH concentration, or when SCOS with high FSH concentration can be predicted on the basis of the pre-operative prognostic data. Unfortunately, no secure pre-operative prognostic elements of sperm recovery exist for NOA patients. Neither the orchidometry and/or the serum FSH concentration are predictive, as also shown by the current data. In this respect, no significant difference was reported in the frequency of positive sperm recovery neither in NOA patients with a bilateral testis volume of 10 ml and FSH concentrations > 20 miu/ml, nor in NOA patients displaying unilateral testis volume > 10 ml or FSH concentrations < 20 miu/ ml (Hibi et al., 2005). Similarly, serum inhibin B concentration in combination with serum FSH concentration is not predictive of the probability of sperm recovery (von Eckardstein et al., 1999). Others demonstrated that sensitivity and specificity were 97% and 83.3% respectively for FSH concentration, 72.2% and 95.5% for serum inhibin B concentration, and 88.9% and 94% for testicular volume (Ziaee et al., 2006). The aim of these surgical techniques is to retrieve spermatozoa to be used for ICSI and, for this reason, sperm viability assessment is essential. Nevertheless, of the above mentioned papers comparing the efficacy of microtese versus TESE: only Schlegel (1999) provide information about sperm viability, reporting a fertilization rate of 65% (95/146) with spermatozoa retrieved by microtese versus 52% (51/98) in standard TESE procedures (P = 0.01). In this study, the eosin nigrosin test was used, when possible, to assess sperm viability on a very tiny aliquot from each biological sample, and showed no significant difference between microtese and TESE samples. Although the ultimate validity of the procedure will be finally assessed based on the subsequent pregnancy outcome, there is no doubt that the highest probability of sperm recovery after microtese gives access to ICSI cycles to a wider range of couples. Future research is aimed at characterizing the recovered spermatozoa and the corresponding ICSI cycle outcome. In conclusion, this randomized controlled study demonstrated that microtese is significantly more effective than TESE in retrieving spermatozoa in NOA patients, especially those having the worse prognosis. As yet, no secure data or examinations are available to predict the retrieval of spermatozoa in NOA patients, microtese is the elective choice at least in patients with high FSH concentrations. References of Urology 163, Dohle GR, Jungwirth A, Colpi GM et al Guidelines on Male Infertility. 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