Hum. Reprod. Advance Access originally published online on February 10, 2006
Human Reproduction 2006 21(6):1551-1554; doi:10.1093/humrep/del012
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How successful is repeat testicular sperm extraction in patients with azoospermia?
1 Clinica Eugin, Calle Entenza, Barcelona, Spain and 2 Centre For Reproductive Medicine, University Hospital, Dutch-speaking Brussels Free University (Vrije Universiteit Brussel), Brussels, Belgium
3 To whom correspondence should be addressed at: Clinica Eugin, Calle Entenza 293-295, 08029 Barcelona, Spain. E-mail: vvernaeve{at}euvitro.com
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Abstract |
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BACKGROUND: Little is known about the extraction rate in repeated sperm retrieval procedures in azoospermic patients. This study aimed to assess the feasibility of repeated sperm recovery in these patients. METHODS: A total of 1066 azoospermic men had their first sperm recovery between 1 January 1995 and 31 December 2003. A total of 381 men had obstructive azoospermia (OA), 628 nonobstructive azoospermia (NOA) and 57 showed hypospermatogenesis. RESULTS: Overall, sperm could be retrieved in all procedures in the 598 cycles performed in OA men (100%). A total of 117, 57, 24, 11, 7 and 1 men underwent, respectively, two, three, four, five, six and seven sperm retrievals; all were successful. Of the 784 procedures performed on the 628 men with NOA, sperm could be retrieved in 384 procedures (49%). During the first testicular sperm extraction (TESE) procedure, sperm could be extracted in 261 men with NOA (41.6%). A total of 103 men had a second attempt, 34 had a third attempt, 11 had a fourth attempt, 6 had a fifth attempt and 2 had a sixth attempt. In these cycles, sperm could be extracted in, respectively, 77 (74.7%), 28 (82.3%), 11 (100%), 5 (83.3%) and 2 (100%) men. CONCLUSION: Repeated TESE ensures a high sperm recovery rate even in patients with NOA. In NOA patients, studies reporting on TESE may therefore overestimate the retrieval rate by reallocating successful patients. These data also show that when no spermatozoa can be obtained after thawing cryopreserved testicular sperm for ICSI in NOA patients, a repeat TESE procedure can be planned.
Key words: azoospermia/repeated TESE/testicular sperm/TESE
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Introduction |
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Since its introduction in 1993 (Craft et al., 1993
; Schoysman et al., 1993), ICSI with testicular sperm has become a routine procedure for patients with azoospermia, whether they suffer from obstructive azoospermia (OA) with normal spermatogenesis or nonobstructive azoospermia (NOA) with testicular failure. In OA, sperm can be retrieved in almost 100% of the cases (Tournaye et al., 1997a). In the population of patients with NOA, however, the possibility of finding sperm is only about 50% (Tournaye et al., 1997a,b). Furthermore, if testicular sperm is found in patients with NOA, the pregnancy rate after one ICSI cycle is low (Vernaeve et al., 2003; Nicopoullos et al., 2004), and repeated ICSI cycles with repeated testicular biopsies are therefore often needed.
Only three studies, based on a limited number of cases, examined the possibility of finding sperm in repeated testicular biopsies in patients with azoospermia (Westlander et al., 2001; Friedler et al., 2002; Kamal et al., 2004). Therefore, we examined whether consecutive sperm retrieval procedures are successful in a large series of patients with azoospermia.
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Materials and methods |
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Patients
In this retrospective case series, patients who had their first testicular sperm extraction (TESE) procedure for ICSI, between January 1995 and December 2003 were included. Patients who had had a previous TESE elsewhere were excluded from the study. All patients in whom sperm was found during a previous biopsy and wishing to undergo a repeat biopsy were accepted, as it is our policy not to repeat the TESE procedure if no spermatozoa were found during a previous trial.
All patients were found to be azoospermic on the basis of at least two semen analyses, including a centrifugation step at high speed. They all had a clinical work up including a physical examination, hormonal assessment (FSH, LH and testosterone) and assessment of biochemical markers. Transrectal ultrasound, karyotype analysis, assessment for Yq microdeletion and analysis of the most frequent cystic fibrosis transmembrane regulator (CFTR) gene mutations were performed in selected patients.
As in the study by Matsumiya et al. (1994), the group of patients with NOA comprised patients with incomplete spermatogenesis. The histology of these patients showed maturation arrest with or without focal spermatogenesis, germ cell aplasia (Sertoli cell-only syndrome) with or without focal spermatogenesis and tubular sclerosis/atrophy. In the case of a mixed histological pathology, the most prominent pattern was used for classification. Patients with a clinical diagnosis of obstruction and revealing normal spermatogenesis were classified as having an OA. Hypospermatogenesis indicates a state of complete but reduced spermatogenic activity and was considered a separate subpopulation. This study was approved by our institutional review board.
Testicular sperm recovery
In patients with a clinical diagnosis of NOA, open excisional testicular biopsies were taken under general anaesthesia or occasionally under loco-regional anaesthesia as described previously by Tournaye et al. (1997a) either on the day of ovum retrieval or during a preliminary surgery with a view to cryopreservation. In patients with OA, biopsies were taken under local anaesthesia. An incision of approximately 1 cm was made through the skin and underlying layers. After incision of the tunica albuginea, gentle pressure was applied to the testicular mass, and a small specimen of the protruding testicular mass was removed using a pair of curved scissors. The weight of each tissue sample was approximately 150 mg. The testicular tissue was placed in a Petri dish containing HEPES-buffered Earle’s medium supplemented with 0.5% human serum albumin (Red Cross, Brussels, Belgium) and taken to the adjacent laboratory. In the laboratory, the wet preparation of testicular tissue was shredded roughly using two microscopic glass slides in a Petri dish (3002; Becton-Dickinson, Aalst, Belgium) on the warmed stage of a stereomicroscope at 40x magnification (Verheyen et al., 1995). During this procedure, the seminiferous tubules were unravelled and broken. The tissue was then further minced with two fine forceps (Lawton, Tuttlingan, Switzerland) in a Petri dish (‘mincing’), until tissue pieces of approximately 1 mm3 or free tubuli pieces of a few millimetres in length were obtained. Under an inverted microscope (400x magnification) the shredded tissue was then checked for the presence of spermatozoa. When spermatozoa or late elongated spermatids (stage Sd2) according to Clermont (1963) were found on wet preparations of the biopsies, surgery was discontinued.
Otherwise, at least four biopsies were taken randomly on each side. Since 1998, enzymatic digestion of the testicular tissue with collagenase type IV has been performed whenever sperm was not found after mechanical shredding (Crabbé et al., 1998). A successful biopsy was defined as the finding of at least one spermatozoon in the biopsy specimen.
During surgery, a single randomly taken biopsy of each testis was sent for histological examination. For histopathology, Bouin’s solution was used as a fixative. The staining was done by using hematoxylin–eosin–safran solution. Given the possible focality of histological patterns, this approach might introduce misclassification in some patients; we nevertheless felt that it would be unethical to send more samples for histological analysis to obtain a more reliable classification.
The testicular cell suspension was frozen for later use, if at least one, preferably motile, sperm was observed after diagnostic retrieval or if, after injection of the mature oocytes at the day of biopsy retrieval, sufficient remaining spermatozoa were supposed to be available for a next ICSI treatment.
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Results |
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A total of 1066 azoospermic men had their first sperm recovery between 1 January 1995 and 31 December 2003. A total of 381 men had an OA, 628 presented a NOA as proven by histopathology. A third category of 57 patients studied showed hypospermatogenesis. The causes of obstruction and spermatogenetic defects are summarized in Table I.
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A total of 78 patients with OA (149 biopsies), 44 with NOA (60 biopsies) and 4 with hypospermatogenesis (6 biopsies) were excluded from the study, because they had their first biopsy before 1995 in 70, 36 and 3 patients, or because they had a previous biopsy elsewhere in eight, six and one patients, respectively, in OA, NOA and hypospermatogenesis.
Overall, sperm could be retrieved in all procedures in the 598 cycles performed in OA men (100%). A total of 117, 57, 24, 11, 7 and 1 patient underwent, respectively, two, three, four, five, six and seven sperm retrievals; all were successful.
In the 784 procedures performed on the 628 men with NOA, sperm could be retrieved in 384 procedures (49%). During the first TESE procedure, sperm could be extracted in 261 men suffering from NOA (41.6%). In repeated procedures, sperm could be extracted in 123 of the 156 TESE procedures in NOA men (78.8%). The extraction rate in the consecutive cycles among patients with NOA is summarized in Table II. Although it is our policy not to repeat a TESE in case of previous failed surgery, two patients who had had a first successful and a second unsuccessful TESE chose to undergo a third one. In both the cases, the third biopsy was also unsuccessful.
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During the first TESE procedure, sperm could be extracted in 140 of the 362 patients with a germ cell aplasia (Sertoli cell-only) (38.7%), in 81 of the 163 patients with a maturation arrest (49.7%) and in 40 of the 103 patients with a tubular sclerosis and atrophy (38.8%). The extraction rate in the consecutive cycles among the different subgroups of patients with NOA is summarized in Table III.
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The average interval between two biopsies in patients with NOA was 10 months (range 2–48). A total of 58 biopsies were performed within an interval of <6 months. Sperm could be retrieved successfully in 48 biopsies (82.7%). In an interval of 
6 months 98 biopsies were performed. Sperm retrieval was successful in 75 procedures (76.5%).
In the 89 surgeries performed among men with hypospermatogenesis, sperm could be extracted in 88 of them (98.9%). In the 57 men who had a first TESE, sperm could be retrieved in all of them. A total of 21 men had a second extraction, which was successful in 20 men (95.2%). Seven men had a third attempt, two had a fourth attempt and one had a fifth attempt; all recovery procedures were successful.
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Discussion |
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Although repeated procedures are often required to obtain the desired child, information regarding the outcome of repetitive TESE procedures is scarce in the literature. Cryopreservation of remaining testicular tissue is a valid option to avoid repeated surgery. However, cryopreservation is not always feasible in NOA, and no sperm will be found after thawing in about 20% of patients; a repeat TESE procedure therefore needs to be performed on the day of oocyte retrieval (Verheyen et al., 2004
). Some couples may also need a repeat procedure to achieve a second pregnancy.
Because the quantity of testicular tissue is limited and some authors have cautioned for possible testicular damage after TESE (Schlegel and Su, 1997; Ron-El et al., 1998), the true prognosis of repetitive TESE is of paramount importance to adequately counsel patients.
In case of OA, it is widely accepted that sperm can be recovered in all patients. Only one study examined the feasibility of repeating testicular sperm aspiration in 22 men suffering from OA (Westlander et al., 2001). They found a sufficient number of spermatozoa to be able to inject all the oocytes in all patients up to the third procedure.
Several studies of patients with NOA found a sperm extraction rate of 30–90% (Friedler et al., 1997; Tournaye et al., 1997a,b; Ezeh et al., 1998; Amer et al., 1999; Schlegel, 1999; Turek et al., 1999; Tsujimura et al., 2002). However, only three small-scale studies examined the feasibility of repeating TESE procedures in patients with primarily testicular failure. The study by Westlander et al. (2001) examined the feasibility of repeating testicular sperm aspiration in 34 nonobstructive azoospermic men. They found that, in one patient, repeating the procedure up to the sixth attempt was feasible. However, their definition of NOA is unclear as no histology was available and may include a substantial proportion of patients with normal spermatogenesis or hypospermatogenesis. The study by Friedler et al. (2002) examined repeated TESE in 22 patients with NOA defined according to histology. They found that repeating the procedure up to the fourth trial was justified. These findings were corroborated by Kamal et al. (2004) with a study of 41 patients with NOA. Repeated TESE trials were successful in 91.5% of patients, if sperm was recovered during the first procedure. Our findings based on a large number of patients, all with a well-defined azoospermia, are in agreement with the previous reported studies.
We may conclude that repeated TESE ensures a high recovery rate even in patients with NOA when a first recovery procedure has been successful. Studies reporting on TESE in NOA patients may therefore overestimate the retrieval rate by reallocating successful patients. These data also show that when no spermatozoa can be obtained after thawing cryopreserved testicular sperm for ICSI in NOA patients, a repeat TESE procedure can be scheduled.
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Acknowledgements |
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We thank the clinical, paramedical and laboratory staff of the Center for Reproductive Medicine. Furthermore, we are very grateful to Mr M.Whitburn of the Language Education Center of our University for proofreading our paper. We also thank Mr Walter Meul for his technical assistance in data retrieval.
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Submitted on August 29, 2005; resubmitted on December 20, 2005; accepted on January 6, 2006.
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