Testicular sperm extraction: impact of testicular histology on outcome, number of biopsies to be performed and optimal time for repetition

Medhat Amer1,2,3, Shawky El Haggar1, Taymour Moustafa1, Taha Abd El-Naser1,2 and Wael Zohdy1

1 Department of Andrology, Cairo University Hospitals and 2 Adam International Clinic, 20 Aden Street, Mohandessin, Giza, Egypt


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Testicular sperm extraction (TESE) may not always be successful in patients with non-obstructive azoospermia, as they only have minute foci of active spermatogenesis from which a tiny number of spermatozoa can be extracted. The aim of this study was to find the percentile incidence of successful TESE in non-obstructive azoospermia patients in relation to various histopathological patterns and the number of performed biopsies, and to determine the optimal time needed for repetition. A total of 216 patients underwent bilateral testicular biopsy taking a single piece from each testis for sperm retrieval and pathological evaluation. In another 100 patients, the same procedure was done but taking multiple samples (maximum four samples/testis). Spermatozoa were successfully retrieved from 37.5 and 49% of patients who supplied single and multiple samples respectively. TESE was significantly higher when multiple samples were taken in all histopathological groups except for Sertoli cell-only syndrome, tubular sclerosis and Klinefelter's pattern. Twenty-seven patients underwent repeated TESE for ICSI between 1 and 24 months from the first procedure; all of them had easy sperm retrieval during the first procedure. Although sperm retrieval was successful in 75 and 94.7% of patients who underwent the second attempt, before and after 3 months respectively, a second TESE was usually more difficult and necessitated multiple sampling.

Key words: azoospermia/sperm retrieval/testicular biopsy/testicular sperm extraction


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is well established that mature testicular spermatozoa could be found in men with non-obstructive azoospermia (Hauser et al., 1994Go; Tournaye et al., 1995Go). Thus, testicular sperm extraction (TESE) combined with intracytoplasmic sperm injection (ICSI) offer azoospermic patients the possibility of fathering their own genetic children even if they do not have normal spermatogenesis. TESE and ICSI have become the standard protocols for treatment of patients with functional azoospermia who have no source of spermatozoa except the testis (Devroey et al., 1995Go). However, TESE may not always be successful in those patients who have only minute foci of active spermatogenesis from which a tiny number of spermatozoa can be extracted (Silber et al., 1995Go). That is why most investigators prefer open biopsies (Friedler et al., 1997Go; Ezeh et al., 1998Go; Rosenlund et al., 1998Go) or biopty gun needle biopsy (Rajfer and Binder, 1989Go; Tuuri et al., 1998; Hovatta et al., 1999) rather than needle aspiration (Craft et al., 1997Go) to maximize chances of finding these rare foci of active spermatogenesis. As there is inconsistency in the literature concerning the pattern of testicular histology in non-obstructive azoospermic men (Hauser et al., 1998Go), attitudes vary concerning the number of testicular samples that should be taken for sperm retrieval in men with non-obstructive azoospermia. Some perform a single testicular biopsy (Silber et al., 1995Go; Verheyen et al., 1995Go) based on the assumption that multi-focal distribution of spermatogenesis throughout the entire testis is present in non-obstructive azoospermia (Silber et al., 1997Go). Others perform and strongly recommend multiple biopsies, since this may enhance the diagnostic accuracy of absolute testicular failure and increase the number of sperm cells retrieved (Tournaye et al., 1996Go, 1997Go; Hauser et al., 1998Go; Ostad et al., 1998Go). If the multiple surgical approach is preferred, there is an increased risk of interruption of the blood supply (Schlegel and Su, 1997Go), post-sampling fibrosis or autoimmune response (Tournaye et al., 1997Go). As the detrimental effect of TESE on spermatogenesis continues for several months, it was advised (Schlegel and Su, 1997Go) not to repeat the procedure on the same testis within 6 months. Testicular sampling evokes two important questions: the optimal number of biopsies that should be performed and the optimum time for repetition. The first aim of this study was to answer these two important questions by determining the percentile incidence of successful TESE in patients with functional azoospermia in relation to various histopathological patterns and to the number of performed biopsies. This is important, as it helps to decide in which histopathological pattern the performance of multiple, rather than a single, testicular sample can be more beneficial and to avoid unnecessary multiple sampling. Our second aim was to evaluate the likelihood of success or failure of second TESE in relation to testicular function and time passed after the first procedure, thus determining the optimal time to repeat TESE procedure if needed.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Two groups (A and B) of patients with azoospermia were recruited from the Adam International Clinic and the Cairo University Andrology Outpatient Clinic.

Group A included 316 patients with functional azoospermia: it included 216 patients (subgroup A1) with a mean age (±SD) of 32.45 ± 6.03 years and duration of infertility ranging from 16 months to 25 years. The mean testicular volume was 12.27 ± 3.07 ml on the left side and 12.55 ± 3.05 ml on the right side. The follicle stimulating hormone (FSH) concentrations ranged from 1 to 68 IU/l with a mean (±SD) of 16.98 ± 11.63 (normal: 2–8 IU/l). They underwent diagnostic bilateral testicular biopsy with a single piece from each testis. One part was subjected to TESE; another part was prepared for ordinary histopathology with paraffin sections. Subgroup A2 included 100 patients with a mean age of 36.43 ± 5.83 and the duration of infertility ranged from 1 to 27 years. The mean testicular volume was 12.73 ± 3.49 ml on the left side and 12.70 ± 3.65 ml on the right side. The FSH concentration ranged from 2.7 to 56 IU/l with a mean of 16.26 ± 11.83 (normal: 2–8 IU/l). They underwent multiple testicular biopsies from each testis for TESE/ICSI. The number of biopsies from each testis varied according to the presence or absence of spermatozoa in the fresh examination and to testicular volume; the number varied from two to four biopsies. We tried to find the positive yield of spermatozoa according to the number of biopsies taken in order to determine the optimum number of biopsies to be taken. No further biopsies were taken once spermatozoa were identified. Two pieces from each biopsied testis (upper and lower pole) were prepared for histopathology.

Group B included 27 patients with functional azoospermia who underwent repeated TESE for ICSI 1–24 months after the first diagnostic procedure. They were classified into three groups according to time passed from the first TESE procedure, which was usually diagnostic. Group B1 underwent repeated TESE during the following 3 months (n = 8 patients). Group B2 underwent repeated TESE after 3–8 months (n = 4 patients). Group B3 underwent repeated TESE after 8–24 months (n = 15 patients). When one or two biopsies were enough to retrieve spermatozoa, we termed it an easy trial. When more than two biopsies were taken, we termed it a difficult trial. As a control group, seven patients with obstructive azoospermia underwent repeated TESE attempts, one patient during the following 3 months, two and four patients after 3–8 and 8–24 months respectively.

Testicular biopsy was obtained under local or general anaesthesia using an open surgical technique described previously (Jow, 1995Go).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
According to histopathological findings, the patients were divided into five groups: early spermatid arrest, primary spermatocyte (C1) arrest, mixed pathology, Sertoli cell-only syndrome (SCO) and Klinefelter's (KF) syndrome pattern and tubular sclerosis. Table IGo shows the incidence of histopathological patterns in both groups. Differences in histopathology between right and left testes were found in 23 and 21.7% of groups A1 and A2 respectively, whereas differences between upper and lower poles of the same testis were found only in 6.6% of all testes of group A2 that underwent multiple biopsies (11/155). Spermatozoa were retrieved from 81 patients in the first group A1 (37.5%) and from 49 patients in the second group A2 (49%). For each histopathological group, multiple biopsies showed a higher percentage of sperm retrieval except in cases with complete SCO, tubular sclerosis and KF pattern (Table IIGo). By taking two biopsies, it was possible to screen 60% of positive cases while three biopsies detected 92%. No more than four biopsies were taken from group A2 (Table IIIGo).


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Table I. Histopathological patterns in biopsy of 316 patients with functional azoospermia: group A1 underwent a single diagnostic bilateral testicular biopsy; group A2 underwent multiple testicular biopsies from each testis
 

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Table II. Percentile incidence of positive testicular sperm extraction (TESE) using single (group A1) and multiple (group A2) testicular biopsies
 

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Table III. Percentile incidence of positive testicular sperm extraction (TESE) according to number of biopsies in patients who had TESE through multiple biopsies (group A2)
 
In group B, the results of the first and second TESE procedure are shown in Tables IV, V and VIGoGoGo. In group B1 (n = 8), spermatozoa were easily retrieved from all patients during the first procedure. During the repeat TESE procedure in these patients, spermatozoa were easily retrieved from five patients (one of them by needle aspiration), retrieved with difficulty in one patient and no spermatozoa could be retrieved in the two remaining patients. In group B2 (n = 4) spermatozoa were easily retrieved from all patients during the first procedure. During repeat TESE, spermatozoa were retrieved easily from three patients and with difficulty from one patient. In group B3 (n = 15) spermatozoa were easily retrieved from all patients during the first procedure. During repeat TESE, spermatozoa were easily retrieved from six patients, with difficulty from eight patients, and no spermatozoa could be retrieved from one patient.


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Table IV. Results of first and second TESE in 27 patients with functional azoospermia who underwent repeated testicular sperm extraction (TESE) for intracytoplasmic sperm injection (ICSI) (group B)
 

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Table V. Results of second testicular sperm extraction (TESE) procedure in patients who had repeated TESE (group B) according to difficulty encountered
 

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Table VI. Comparison of the results of the second testicular sperm extraction (TESE) when performed before and after 3 months following the first biopsy
 
During repetition, spermatozoa were retrieved in 75% of the first group, 100% of the second group and in 93.3% in the third group (Table VGo). In 10 patients TESE was difficult (i.e. more than two biopsies; Table VGo). In three patients no spermatozoa could be retrieved even after 14 biopsies in one of them. Two of them underwent repeated TESE in less than 3 months and the third repeated the procedure after 8 months (Table VIGo). All the 13 patients with difficult or failed retrieval at the second TESE had had easy sperm retrieval during the first attempt. The seven control cases with obstructive azoospermia had easy sperm retrieval during the first and repeated TESE.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The advent of ICSI with testicular spermatozoa has revolutionized the treatment options available to men with non-obstructive azoospermia. However, failure of TESE may occur in up to 57% of attempts (Devroey et al., 1995Go; Kahraman et al., 1996Go; Friedler et al., 1997Go; Schlegel et al., 1997Go; Rosenlund et al., 1998Go). The alternative use of spermatids for oocyte injection is rather disappointing (Amer et al., 1997Go; Vanderzwalmen et al., 1997Go). So it is of utmost importance to use the most reliable and safe method available in order to detect and retrieve testicular spermatozoa in patients with non-obstructive azoospermia.

Several approaches have been described for extraction of the rare spermatozoa present in the testes of men with complete azoospermia and limited sperm production. Some groups utilize fine needle aspiration (Turek and Ljung, 1997Go). This technique provides diagnostic information and a reliable sperm yield in obstructive azoospermia with a minimally invasive technique (Craft et al., 1997Go). However, the number of spermatozoa retrieved is quantitatively limited (Syenkin et al., 1998Go). Several controlled studies comparing fine needle aspiration with open testicular biopsy for men with non-obstructive azoospermia were carried out. These studies have demonstrated that open biopsies are required to achieve an optimal chance of finding the rare foci of testicular spermatozoa present within these poorly functioning gonads (Friedler et al., 1997Go; Ezeh et al., 1998Go; Rosenlund et al., 1998Go). Other studies have adopted the use of biopty gun needle biopsy in patients with non-obstructive azoospermia because the needle reaches a wider area of testicular tissue and so avoids the possibility that the biopsy sample is taken from a small area locally devoid of spermatogenesis (Rajfer and Binder, 1989Go; Hovatta et al., 1995Go; Tuuri et al., 1999). This technique is simpler and cheaper than open biopsy and can be carried out under local anaesthesia. Although it was considered (Tuuri et al., 1998) as the optimal method for the retrieval of testicular spermatozoa giving sufficient amount of tissues and spermatozoa for ICSI, cryopreservation and histopathological evaluation, colour Doppler analysis was not performed to verify the safety of this blind technique against the open technique.

It has been claimed (Silber et al., 1997Go) that the distribution of spermatogenesis in the testicles of azoospermic men is homogeneous. It was suggested that a single testicular biopsy is sufficient to diagnose the presence of spermatozoa based on the assumption that multi-focal distribution of spermatogenesis throughout the entire testes is present in non-obstructive azoospermia. When comparing our results of single versus multiple testicular biopsies in the same histopathological groups, we found significant improvement in the percentage of successful TESE with multiple biopsies. Our results showed successful TESE in 85% of patients with spermatid arrest using multiple biopsies versus 58.7% with single biopsy, 44% in C1 arrest versus 27.7% with single biopsy and 65.4% in patients with mixed pathology versus 56.6% with single biopsy. However, there was no significant difference in cases with complete SCO, tubular sclerosis and KF where a single biopsy proved to be sufficient and multiple biopsies were not necessary. Knowing the exact histopathological diagnosis from a previous report can help in selecting candidates for multiple biopsies and avoid unnecessary surgical trauma in others. Another important advantage is that it can predict sperm retrieval success with TESE (Tournaye et al., 1997Go; Ezeh et al., 1999Go; Su et al., 1999Go). Our results confirm this, as sperm retrieval incidence was proportional to spermatogenic activity in the examined histological samples.

Hauser et al. recommended that at least three open testicular biopsies from each testis were needed in order to reach the most accurate diagnosis in cases of absolute testicular failure (Hauser et al., 1998Go). Tournaye et al. practised extensive multiple biopsies until spermatozoa are found or when the whole testicular mass is bilaterally sampled at random (Tournaye et al., 1998). Ostad et al. did not stop their biopsies under optical magnification until either sperm cells were found or no avascular regions of the testes were left (Ostad et al., 1998Go). In our study, we found that the performance of three testicular biopsies will screen the majority of cases, confirming the results of Hauser et al. (Hauser et al., 1998Go). However, when we reached our maximum of four quadrant biopsies, spermatozoa were retrieved from four more cases. In group B, the number of biopsies taken to retrieve spermatozoa from 10 patients varied from three up to 16 biopsies from both testes although previous sperm recovery was successful after a maximum of two biopsies in the first attempt. There is no doubt that performing multiple biopsies can save ICSI cycles, although it lengthens operation time compared to single biopsy and may exert an increased complication rate. Schlegel and Su elegantly described the consequences of multiple TESE procedures, which may even cause complete testicular devascularization (Schlegel and Su, 1997Go). The introduction of microsurgical TESE, besides increasing the yield of TESE, may alleviate the potential hazards of multiple biopsies (Schlegel, 1999Go).

Limiting testicular biopsy for ICSI to those with a high chance of having testicular spermatozoa has not been possible because of the poor predictive value of current clinical and laboratory methods (Kahraman et al., 1996Go; Tournaye et al., 1996Go, 1997Go). Tournaye et al. suggested that a preliminary testicular biopsy might be preferable in patients suffering from secretory azoospermia in order to guarantee sperm retrieval for the planned TESE-ICSI attempt (Tournaye et al., 1997Go). However, Vanderzwalmen et al. showed that in some patients with obstructive 3/115 (2.6%) and non-obstructive 42/137 (30.6%) azoospermia, no spermatozoa could be found in different testicular samples at the time of the TESE–ICSI attempt, notwithstanding previous positive biopsies (Vanderzwalmen et al., 1997Go). Moreover, even when the testes appear clinically normal after the operation, repeated or multiple needle or open testicular biopsies are required for diagnosis and TESE could subject the patient to potential risks of vascular injuries (Harrington et al., 1996Go; Schlegel and Su, 1997Go). Furthermore, spermatogenesis is an intricate and delicate process easily influenced by its environment. Although normal men produce 8–10x107 spermatozoa/day, men with functional azoospermia rarely produce any spermatozoa. Therefore, any adverse effect on testicular function for a man with functional azoospermia will be likely to ablate the marginal level of sperm production present (Schlegel and Su, 1997Go). Although in our study we did not have any problem in retrieving spermatozoa for a second attempt in obstructive azoospermia (7/7), all patients with difficult 10/27 (37%) or no sperm retrieval 3/27 (11.1%) had functional azoospermia and severe spermatogenic defects in their testicular histopathology. Finding an alternative method other than testicular biopsy to predict successful sperm retrieval will certainly be an important achievement in order to avoid the hazards of repetition in the vulnerable non-obstructive azoospermia group.

Patients with non-obstructive azoospermia have minute foci of active spermatogenesis from which spermatozoa could be retrieved; the fear is that the first biopsy may hit the only area of active spermatogenesis, which could be removed with the first biopsy. Silber et al. thought that this might not be a serious problem because the distribution of a minute quantity of spermatogenesis in non-obstructive azoospermia is diffusely multi-focal throughout the whole testis (Silber et al., 1997Go). We do not agree with this argument because we failed in three of our patients to retrieve testicular spermatozoa in a second TESE. Those patients should be counselled about this unhappy expectation.

Schlegel and Su had demonstrated the possible bad effects of TESE on testicular tissue and reported that 82% of patients develop ultrasonographic abnormalities in the testis up to 6 months and impaired testicular blood supply may occur following TESE with multiple biopsies in patients with functional azoospermia (Schlegel and Su, 1997Go). They also reported that patients who underwent repeat TESE before 6 months of the first procedure are more likely to have no spermatozoa in their biopsies (75%), while patients who underwent repeated TESE after 6 months had successful sperm retrieval in 80% of them. Thus they recommended repeating TESE 6 months or longer from the first procedure. In our experience, spermatozoa were retrievable during repeat TESE in 75% of men when performed before 3 months and in 94.7% when performed after 3 months (Table VIGo; P > 0.05). Although the differences were not statistically significant, most probably due to the small number of cases, we believe that TESE should never be repeated before an interval of 3 months. Waiting for 6 months or even up to 2 years will not necessarily provide an easy procedure with a successful outcome.

Hauser et al. showed that the performance of multiple biopsies is recommended in all cases of functional azoospermia: in terms of the presence of spermatozoa, no special site was recommended as the best location for a single retrieval. In our work, comparing between upper and lower pole biopsies, we did not find any difference in sperm prevalence, although histopathological differences were present in 6% of cases. In a trial to predict the site of active spermatogenesis (Foresta et al., 1998Go), the use of colour Doppler of the testis was recommended to evaluate intratesticular blood vessel distribution before TESE/ICSI in 16 patients with non-obstructive azoospermia. By this method, fine needle aspiration cytology (FNAC) was performed in testicular regions showing the presence of blood vessels. In these selected areas, sperm retrieval was possible in 12 out of 16 of cases compared with extraction of only Sertoli cells when FNAC was done in the middle portion of the testis in the same patients.

Our findings imply that a single biopsy is sufficient in documented cases of SCO, tubular sclerosis and KF pattern; otherwise, multiple biopsies are indicated. Although patients with normal testicular histology have no problem in the second retrieval even before 3 months, 3 months is the minimum time to elapse before repeating TESE in non-obstructive azoospermia with success. A second TESE may be difficult or even fail in non-obstructive azoospermia. To avoid this unhappy event, we recommend that it is better to substitute diagnostic biopsy with other non-invasive methods such as detecting spermatids in the ejaculate, as an alternative index of successful sperm retrieval prior to TESE (Ezeh et al., 1998Go). An ideal solution is to plan an ICSI trial with the biopsy first to have the best chance of using fresh spermatozoa, if present, and to reduce waste of time and money if no spermatozoa are retrieved. An ICSI trial with biopsy first means preparing the wife for ICSI and, a few hours before oocyte retrieval, the biopsy is taken and extensively processed for the presence of spermatozoa; if no spermatozoa are retrievable, ovum retrieval is cancelled. We are currently studying the use of a surgical microscope to avoid subtunical vessels and to select healthy seminiferous tubules based on tubular diameter as recently advised (Schlegel, 1999Go) to minimize the risk of arterial injury and decrease tissue loss.


    Acknowledgments
 
The authors wish to thank the clinical, paramedical and laboratory staff of Adam International Clinic, and especially colleagues in the operating theatre, and the IVF laboratory. We are also very grateful to Professor Mahmoud Shaker Abd El Aziz, of the language education centre of Cairo University, for correcting this manuscript.


    Notes
 
3 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on May 24, 1999;