Assisted reproduction for the treatment of azoospermia

Transcription

1 Assisted reproduction for the treatment of azoospermia Anat Safran 1, Benjamin E.Reubinoff, Anat Porat-Katz, Joseph G.Schenker and Aby Lewin IVF Unit, Department of Obstetrics and Gynecology, Hadassah Hebrew University Medical School, Ein-Kerem, P.O.Box 12000, Jerusalem IL-91120, Israel 'To whom correspondence should be addressed Azoospermia, the most severe form of male infertility, is caused by obstructions in the genital tract or by testicular failure. Microsurgical techniques are available for the correction of some of these obstructions but no effective treatment is available for testicular failure. In recent years, methods have been developed for direct surgical sperm sampling from either the epididymis or the testis to be used by intracytoplasmic sperm injection. The main approach proven to be effective for the retrieval of spermatozoa from the epididymis in patients with obstructive azoospermia is microsurgical epididymal sperm aspiration, although recently the retrieval of spermatozoa by fine needle aspiration was shown to be equally effective. Recovery of spermatozoa is also now performed in patients with severely deficient spermatogenesis using testicular open biopsy as well as aspiration by fine needle. The ultimate choice of sperm retrieval method in these patients will depend not only on sperm availability, but also on the physiological consequences of the different techiques on testicular function. This article summarizes the recent advances achieved in the treatment of azoospermic patients using these assisted reproduction surgical techniques. Key words: ICSI/IVF/male factor infertility/sperm aspiration Introduction Male factor is one of the most frequent causes of infertility and presents a variety of endocrine, genetic and immunological aetiologies, as well as sexual malfunction, varicocele and genital infections. The most severe form of male infertility is azoospermia, a term originating from the Greek words azoos (lifeless) and sperma (seed). Thus, azoospermia defines the condition of absence of spermatozoa in the semen and is due to either obstruction in the genital tract or testicular failure. In obstructive azoospermia the process of spermatogenesis is not impaired. The most common causes of obstruction are epididymovasal occlusions caused by infections, congenital bilateral absence of the vas deferens (CBAVD) and ligation or resection of the vas deferens during hernia repair, prostatic and vesicle surgery or vasectomy. Several microsurgical techniques are Human Reproduction Volume 13 Supplement European Society for Human Reproduction and Embryology 47

2 A.Safran et al. available for surgical correction of these obstructions. Epididymovasostomy, the main procedure performed for the correction of post-infection obstruction, was shown to provide 32-47% success in achieving patency. Vasovasostomy is performed mainly for reversal of vasectomy and patency was reported to be achieved in 30-88% of patients with a pregnancy rate of 18-53%. The major advantage of successful microsurgical intervention is the possibility of achieving multiple pregnancies following a single operation. Thus, when the operative skills are available, microsurgery is offered to patients for reversal of vasectomy and for selected cases with epididymovasal occlusions. With the new advances in assisted reproductive techniques, patients with poor surgical prognosis can now be referred to surgical procedures of sperm aspiration to be used for in-vitro fertilization (IVF). Non-obstructive azoospermia is defined as a condition in which there is an impairment of various degrees in the process of spermatogenesis. For patients with testicular failure (with the exception of hypogonadotropic hypogonadism), no corrective approach is available, since all attempts at medical treatment failed. The condition of hypogonadotrophic hypogonadism is caused by pituitary or hypothalamic deficiency and treatment includes hormonal supplementation for the duration of time required for resumption of spermatogenesis. Thus, the only approach for treatment for non-hypogonadotrophic, non-obstructive, azoospermic patients is a surgical intratesticular attempt for the retrieval of spermatozoa. The correct differential diagnosis of obstructive and non-obstructive azoospermia is important for the implementation of proper treatment, prognosis and counselling. Traditional clinical criteria defining these two groups are summarized in Table I. Nevertheless, in some cases, a poor correlation was found between the preoperative clinical features and testicular histology. Furthermore, recent surgical attempts to recover spermatozoa in cases of apparent testicular failure have proved successful in many cases, thus further reducing the prognostic value of the clinical parameters. Final prognosis of sperm availability in azoospermic patients can therefore be achieved only with the combination of the clinical parameters and direct surgical sperm sampling. This article summarizes the advances achieved during recent years in the treatment of patients with both obstructive as well as non-obstructive azoospermia using assisted reproductive techniques. Sperm retrieval methods for the treatment of obstructive azoospermia The first azoospermic patients to be treated by assisted reproduction techniques were those with obstructive azoospermia. Microsurgical epididymal sperm aspiration (MESA) combined with IVF has led to the achievement of viable pregnancies (Temple Smith et al., 1985; Silber et al., 1990) but was found to offer limited success primarily due to low fertilization and implantation rates (Hirsh et al., 1994). The introduction of intracytoplasmic sperm injection (ICSI) by Palermo et al. (1992) has opened new horizons for the treatment of these 48

3 Table I. Clinical features related to obstructive and non-obstructive azoospermia Treatment of azoospermia Obstructive (excretory) Patient's history Epididymitis Hernia repair Prostatic or bladder surgery Cystic fibrosis Vasectomy Neurogenic disorders: Retrograde ejaculation Impaired contractility of vas and vesicles in diabetes following lymph node dissection (testicular tumour) Serum follicle stimulating hormone Usually normal Physical examination Normal testicular size Scrotal testicular location Possible absence of vas Possible induration of epididymis Seminal markers Normal/absent/low fructose (seminal vesicles) 13 and low carnitine (epididymis) Testicular biopsy Normal spermatogenesis Non-obstructive Cryptorchidism a Mumps orchitis Torsion Trauma Chemotherapy Irradiation Elevated/normal Low in hypogonadotropic hypogonadism Small/normal testicular size Scrotal testicular location/cryptorchidism Gynaecomastia in Klinefelter Normal fructose and carnitine Germinal hypoplasia (hypospermatogenesis) Spermatogenic arrest (maturation arrest) Germinal aplasia (Sertoli cells only) Tubular hyalinization Testicular fibrosis a Cryptorchidism may cause irreversible testicular damage and azoospermia, depending on the timing and efficiency of medical (human chorionic gonadotrophin) and surgical treatment. b Fructose is absent or low in some patients with congenital absence of the vas deferens and in some patients with obstructions of the ejaculatory ducts. patients. The efficacy of combining MESA with ICSI for the treatment of infertility due to CBAVD was analysed (Tournaye et al., 1994), demonstrating high fertilization (58%) and pregnancy (35.7%) rates even with the use of grossly impaired epididymal spermatozoa. MESA is generally performed under general anaesthesia. After unilateral hemiscrototomy, the epididymis is explored using an operating microscope and carefully dissected to open several epididymal tubules by microscissors. The epididymal fluid is aspirated using a glass pipette and transferred into a conical test tube. Sperm preparation usually includes separation on a two-layer Percoll gradient. A randomized comparative study aimed to determine whether ICSI could achieve better results than conventional IVF with microsurgical aspiration of spermatozoa in patients with CBAVD or 49

4 A.Safran et al. irreparable obstructions (Silber et al., 1994). Results showed high fertilization rates and the development of normal embryos in 82% of MESA/ICSI cycles as compared to 19% with conventional IVF. Overall fertilization and pregnancy rates were significantly higher using ICSI, being 45% and 47% as compared to 6.9% and 4.5% for conventional IVF, respectively. It was thus suggested that ICSI may be mandated for all future MESA patients. It has been suggested that the aetiology of obstruction may affect treatment results. Indeed, in a recent study by Mansour et al. (1997), a significant difference in pregnancy rate was observed when epididymal spermatozoa were obtained from patients with CB AVD (20.4%) as compared with patients without genetic cause of obstruction (37%). The striking finding in the retrieval of spermatozoa from the chronically obstructed epididymis was the inversion of the expected pattern of motility, usually best in the most proximal region (Silber et al., 1990). Furthermore, a remarkably high pregnancy wastage was reported by Tournaye et al. (1994), who retrieved spermatozoa mainly from the cauda. The observation that sperm cells from a more proximal region of the epididymis provide high fertilization rates by ICSI may indicate that although passage through the epididymis is required for normal fertilization, it is not a prerequisite for a sperm cell to gain its intrinsic ability to fertilize the egg and result in normal embryo development once penetration is accomplished by ICSI. The combination of MESA/ICSI also enabled the freezing of epididymal spermatozoa to be used successfully in additional ICSI cycles (Devroey et al., 1995b) avoiding repeated scrotal surgery. In a recent study by Oates et al. (1996), frozen-thawed epididymal spermatozoa were shown to lead to equivalent fertilization and pregnancy rates as compared with fresh epididymal spermatozoa. Based on these results, a new approach was suggested in which sperm and oocyte harvesting need not be performed simultaneously. This scheme may ease the burden of partner scheduling and assure the availability of spermatozoa prior to ovulation induction, thus avoiding unnecessary ovarian stimulation and oocyte retrieval. In some patients requiring MESA for obstructive azoospermia, often no spermatozoa can be retrieved from the epididymis due to extensive scarring from previous corrective microsurgery or MESA procedures, or due to hypoplasia. In some cases the epididymis on both sides is completely absent. In these patients, the only source of spermatozoa is within the testicles. The fertilizing ability of testicular spermatozoa obtained by open testicular biopsy was demonstrated for the first time in a patient in which epididymal aspiration failed (Craft et al, 1993). The first pregnancy following the use of testicular spermatozoa obtained by testicular biopsy in a patient in which epididymal sperm retrieval failed (Schoysman et al., 1993), offered a new opportunity for patients with inoperable excretory occlusion. In a small series described by Devroey et al. (1994), the association of testicular sperm extraction (TESE) and ICSI yielded a high fertilization rate in males with absence of the epididymis. In a larger series of obstructive patients, a study was undertaken to compare the results obtained using epididymal spermatozoa to those obtained using testicular spermatozoa when epididymal aspiration failed or when the epididymis was absent or severely 50

5 Treatment of azoospermia scarred (Silber et al, 1995c). The objective of this study was to determine whether ICSI could salvage the most severe MESA cases where there were absolutely no epididymal spermatozoa available, by resorting to TESE. The procedure is usually performed under general anaesthesia. An incision is made in the scrotal skin and carried through the peritoneal tunica vaginalis. A small incision is then made in the tunica albuginea and the small portion of extruding testicular tissue is excised and minced in HEPES-buffered medium. The effluent is centrifuged and the pellet resuspended in medium and kept in the incubator until it is used. It was demonstrated in the above study as well as by others (Mansour et al, 1997) that in obstructive azoospermia, spermatozoa of both epididymal and testicular origin yield similar fertilization, cleavage and ongoing pregnancy rates using ICSI, although the main disadvantage of testicular spermatozoa was the low number of sperm cells retrieved and their poor motility, making cryopreservation of these cells more problematic than with epididymal spermatozoa (Silber et al., 1995c). In another study (Nagy et al., 1995), the poor parameters of testicular spermatozoa were compared to epididymal and ejaculated spermatozoa. The mean total free sperm count of testicular spermatozoa was 0.54X 10 6 as compared to 46.2X 10 6 and 17.8X 10 6 for epididymal and ejaculated spermatozoa, respectively. Testicular sperm always showed only a slow and sluggish local motility, but the number of motile spermatozoa slightly increased after 1-2 h of in-vitro incubation. It was thus suggested that in cases of obstructive azoospermia, the MESA/ICSI technique should be attempted first, allowing multiple aliquots to be frozen and used in subsequent cycles. In cases where epididymal spermatozoa are not retrievable, testicular biopsy could be a final resort that is also very effective, providing most patients with embryos for transfer (Abuzeid et al., 1995; Mansour et al., 1996). Despite the lower number of sperm cells retrieved from the testis and their extremely poor motility, the fertilizing ability of these cells was shown to be retained following cryopreservation, as pregnancies were achieved following the injection of cryopreserved spermatozoa recovered from testicular biopsies in cases of obstructive azoospermia (Gil-Salom et al, 1996; Podsiadly et al, 1996). Consequently, successful sperm aspiration for the treatment of obstructive azoospermia was reported using a less invasive approach of sperm retrieval from the epididymis by fine needle aspiration. In the first study (Shrivastav et al, 1994), percutaneous epididymal sperm aspiration (PESA) was applied for the treatment of seven patients with obstructive azoospermia. The procedure is performed either under general or local anaesthesia and the aspiration apparatus includes a 25-gauge butterfly needle connected to a 1 ml tuberculin syringe. The needle is directly inserted into the epididymis, suction is applied and the aspirates are washed into a sterile tube. Spermatozoa are usually prepared by a discontinuous Percoll gradient. In the study by Shrivastav et al (1994), high sperm counts were achieved in all cases ( XlO 6 /ml) and three pregnancies were established. In a larger series (Tsirigotis et al, 1996), a high recovery rate (85.5%) of spermatozoa from the epididymis using the percutaneous aspiration approach, as well as high fertilization (52.6%) and pregnancy (33.3%) rates were 51

6 A.Safran et al. reported. These results were comparable to those achieved applying MESA for the treatment of obstructive azoospermic patients (Silber et al., 1994). The quality of spermatozoa retrieved by PESA varied widely and it was suggested that ICSI should be used to maximize fertilization rates (Craft et al., 1995a). In light of these studies, it was concluded that the PESA technique is simple, efficient and does not require specific microsurgical skills. It was also suggested to be associated with fewer complications than an open microsurgical operation (Craft et al., 1995a,b). A similar approach of percutaneous sperm aspiration from the testis (TESA; Craft and Tsirigotis, 1995) evolved from the PESA procedure. It was suggested that TESA be applied to men with active spermatogenesis, with or without an obstruction, in situations where spermatozoa are not recovered following MESA/PESA operations and for some non-obstructive cases of azoospermia. The advantages of the percutaneous approach raised in relation to PESA were relevant for the TESA technique as well, especially in view of the fact that it does not require open surgery and removal of a large volume of tissue. With the view of circumventing the need for open surgical interventions, we have also adopted the percutaneous approach combining testicular and epididymal fine needle aspiration (TEFNA) for the treatment of patients with obstructive azoospermia (A.Safran et al., unpublished data). The technique is performed in a similar manner to previous publications (Shrivastav et al., 1994; Craft et al., 1995b), with some modifications. The aspiration set-up includes a 23-gauge butterfly needle connected to a 20 ml syringe installed in an aspiration handle for the creation of steady suction. The needle is introduced into the caput of the epididymis and suction is established by using the aspiration handle device. Following each aspiration, when epididymal fluid ceases to accumulate in the tube, the needle is withdrawn and the aspirate is washed into a 4-well plate containing HEPES-buffered medium. Sperm is prepared using a two-layer Percoll gradient. Eleven patients underwent 12 treatment cycles. In 11 cycles, sperm cells were easily recovered from the caput region of the epididymis and only one or two punctures were needed. For one patient, spermatozoa could not be retrieved from the epididymis as a result of severe scarring following previous MESA operations. Spermatozoa were recovered by fine needle aspiration from the testes using the same set-up. Mature sperm cells were available in all 12 cycles and five clinical pregnancies (41%) were achieved. In 11 cycles, spare spermatozoa were cryopreserved. No complications were noted in any of these patients. We thus concluded that by employing this relatively simple, inexpensive and well-tolerated technique, sperm recovery can be predicted for almost all patients with obstructive azoospermia. If spermatozoa are not retrieved from the epididymis, TEFNA can immediately be performed using exactly the same set-up. Concern has been expressed as to whether blind needle puncture of the epididymis or the testis can cause damage and post-operative complications such as infection and haematoma. In an animal model, a testicular atrophy rate of 5% has been reported following percutaneous testicular puncture (Goldstein et al., 1983). It is likely that these adverse effects were a consequence of damage to the testicular vasculature in the spermatic cord. Such risks are significantly 52

7 Treatment of azoospermia reduced in humans because of the use of fine needles, the size of the testis and the fact that puncture of both the epididymis and the testis is performed away from the main vascular supply (Craft and Tsirigotis, 1995). Indeed, post-operative complications of patients undergoing these procedures were minimal, with few patients complaining of short-term mild pain. Despite the simplicity of the TESA method, it was still suggested that PESA should be the primary aspiration technique for men with obstructions, because of the ability to recover larger numbers of motile spermatozoa from the epididymis, some of which can be cryopreserved for future use (Tsirigotis and Craft, 1996). There seem to be differences in the number of sperm cells obtained by the two sperm retrieval techniques in obstructive azoospermic patients, with the recovery of fewer spermatozoa using the percutaneous approach. The number of spermatozoa retrieved by TEFNA is in the order of hundreds of thousands to a few millions. Since only a few dozen motile spermatozoa are required per treatment cycle, the number of spermatozoa retrieved using TEFNA enabled their use in all treatment cycles as well as their cryopreservation for several future attempts in most cases. A few other reports have been published in which attempts were made to avoid the need of large tissue sampling for the treatment of obstructive azoospermia. A modified percutaneous sperm aspiration technique was described by Cha et al. (1997). A small incision is made in the scrotum, creating a direct view of the epididymis. Sperm cells are aspirated using a 24-gauge angio-needle and sperm preparation is performed as in the MESA approach. High fertilization (77.3%) and pregnancy (43.5%) rates were achieved using this technique and it was suggested that making this small opening may reduce the trauma caused by blind puncture of the epididymis. Hovatta et al. (1995) described an alternative technique of testicular sperm retrieval by using a biopsy gun needle. Using this approach, small pieces of testicular tissue could be sampled from a wider area of the testis and spermatozoa were obtained in 82% of cycles. Bourne et al. (1995) reported successful aspiration of testicular spermatozoa by a 20-gauge Menghini biopsy needle. Therefore, it seems that simple percutaneous aspiration or needle biopsy (Watkins et al., 1997) offers a high sperm recovery rate while being easy to perform, well tolerated by the patients and cost effective. Sperm retrieval methods for the treatment of non-obstructive azoospermia In non-obstructive azoospermia, the process of spermatogenesis is impaired, the epididymis is usually devoid of spermatozoa, and in some of these patients only a few foci with complete spermatogenesis may exist in the testicles (Silber et ah, 1995b). Initially, few case reports were published in which successful sperm recovery by TESE was achieved in patients with testicular failure. Yemini et al. (1995) reported fertilization following the injection of few spermatozoa obtained by open testicular biopsy in an azoospermic patient with almost complete 53

8 A.Safran et al. spermatogenic arrest and testicular tubular atrophy. In another study, an azoospermic patient with small testes, high levels of serum follicle stimulating hormone (FSH) and testicular histology of partial germinal aplasia with focal spermatogenesis was treated by TESE (Gil-Salom et al, 1995). A few spermatozoa with 'twitching' motility were recovered, leading to a high fertilization rate (73%) and a pregnancy. In this case report it was demonstrated for the first time that patients with almost complete spermatogenic failure and markedly elevated FSH levels may be successfully treated by TESE combined with ICSI. Concomitantly with these studies, the efficacy of ICSI with TESE in a series of 15 patients with non-obstructive azoospermia was analysed (Devroey et al, 1995a). In this study, the inclusion of patients suffering from non-obstructive azoospermia was mainly based on high FSH concentrations and small testicular size. Consequently, the histology of these patients revealed severe spermatogenic defects in most cases. A high sperm recovery rate (86%) was reported, and the fertilization (47.8%) and pregnancy (25%) rates achieved were remarkably similar to those obtained using testicular spermatozoa from patients with normal spermatogenesis (Devroey et al., 1994). Further experience of several groups with TESE and ICSI for the treatment of non-obstructive azoospermic patients has been recently published (Tournaye et al., 1995, 1996a; Devroey et al., 1996; Kahraman et al., 1996a,b; Silber et al, 1996; Friedler et al, 1997). From all these studies few important conclusions could be drawn. It was suggested that an attempt of sperm recovery should be offered to all azoospermic patients, irrespective of serum FSH levels, testicular size or medical history (Tournaye et al, 1995; Mansour et al, 1997). When the outcome of treatment was analysed in relation to testicular histology, no correlation was found between histological diagnosis and sperm recovery in the wet preparation (Tournaye et al, 1996a). This pointed out the shortcoming of a single testicular specimen in making the correct histological diagnosis and could be explained by the focal nature of spermatogenesis in these patients. It was also demonstrated that the normal fertilization rate was significantly reduced in oocytes injected with spermatozoa deriving from men with germ-cell aplasia and maturation arrest (Tournaye et al, 1996b). However, once normal fertilization was achieved, embryonic development was comparable with other infertility aetiologies. The fertilizing ability of sperm cells recovered from cryopreserved testicular tissue obtained by TESE, which led to the achievement of viable pregnancies from non-obstructive azoospermic patients, was recently demonstrated (Oates et al, 1997). Because of the difficulty in predicting the availability of spermatozoa in these patients, it was suggested in this study that sperm recovery should be performed prior to initiation of an ICSI cycle. If spermatozoa are found, the tissue can be cryopreserved and become the 'sperm source' for multiple ICSI cycles. This approach could prevent surgical intervention in the female partner when sperm recovery fails. Histological analysis of patients with non-obstructive azoospermia has demonstrated that if the entire testis is carefully sampled, occasional foci of complete spermatogenesis will be found in more than half of cases (Silber et al, 1995a,b, 1996). It should however be emphasized that in a recent study by Silber et al 54

9 Treatment of azoospermia (1997b), the distribution of active areas of spermatogenesis in cases of testicular failure was suggested to be evenly scattered throughout the testicle, rather than in a few patchy areas. Nevertheless, the concept of spermatogenesis being confined to patchy areas in non-obstructive azoospermic patients was the basis for a study that we undertook in order to evaluate the feasibility of obtaining spermatozoa by multiple sampling of the entire testis by TEFNA in an azoospermic patient with maturation arrest (Lewin et al., 1996). Eight sperm cells with apparently normal morphology and signs of motility were found and one oocyte was normally fertilized out of four mature oocytes injected. A single embryo was transferred and a pregnancy and delivery of a healthy boy were achieved. We postulate that unlike a single testicular biopsy, in cases of testicular failure the TEFNA approach may enable the surgeon to reach multiple areas and thus increase the chance of 'hitting' a rare site of active spermatogenesis. Based on this hypothesis, we initiated a prospective study in which epididymal and testicular fine needle aspiration was applied to all non-obstructive azoospermic patients (Lewin, A., Safran, A., Reubinoff, B.P. et al. unpublished data, 1998). Confirmation of the non-obstructive nature of azoospermia, according to the classification by Levin (1979), was made from the histological report of either the diagnostic preoperative biopsy, or from tissue sampling during the sperm retrieval procedure. Pseudo azoospermia was excluded from this group, as all men submitted ejaculate samples on two occasions prior to surgery and these samples underwent extensive examination after high velocity centrifugation and were all found to be devoid of any spermatozoa. Of 85 patients, spermatozoa could be recovered in 50 (58.8%) and a pregnancy rate per transfer of 42.8% was achieved. A low rate of mild complications was recorded in this group as only three patients suffered pain necessitating treatment with oral analgesia. In summary, a high sperm recovery rate could be achieved applying TEFNA for the treatment of non-obstructive azoospermic patients and, once spermatozoa were retrieved, the outcome was comparable to that achieved by other sperm retrieval methods. With regard to the dispute concerning the distribution of spermatogenic sites in the testicles of non-obstructive azoospermic patients, our experience supports the hypothesis of focal spermatogenesis because, in many cases during the first search of the wet preparation, spermatozoa could be detected in one out of a few tens of aspiration samples. Spermatozoa could be recovered even in four of six cases of Klinefelter syndrome and a delivery following preimplantation diagnosis and the transfer of a normal 46XY embryo was achieved (Reubinoff et al., 1998). Sperm recovery from non-mosaic 47XXY Klinefelter patients using TESE was also reported (Tournaye et al., 1996b) as well as the constitution of sex chromosomes in the embryos obtained from three of these patients (Staessen et al., 1996). The conception achieved in our centre using TEFNA (Reubinoff et al., 1998), is among the first few pregnancies (Staessen et al., 1997) obtained so far using spermatozoa aspirated from azoospermic non-mosaic Klinefelter individuals. We thus suggest that TEFNA may be considered as an alternative approach to TESE for the treatment of nonobstructive azoospermic patients. 55

10 A.Safran et al. Recently, the efficiency of testicular sperm retrieval by TEFNA was compared with open biopsy and TESE "in 37 rigorously selected patients with nonobstructive azoospermia (Friedler et al., 1997). All patients underwent both retrieval techniques, thus each patient served as his own control. Whereas by TEFNA, spermatozoa enabling the performance of ICSI were found in four patients out of 37 (11%), open biopsy and TESE yielded spermatozoa in 16 cases (43%). Several explanations could be given for the discrepancy in the rate of sperm retrieval achieved in this study as compared to ours, applying TEFNA for patients with proven testicular failure. In our protocol, the search for spermatozoa, in most patients, required 5-8 h and in many cases only few spermatozoa were found after several hours. If the two techniques, TEFNA and TESE, are performed sequentially on the same day, it is probably impossible to invest the same amount of time for the search of spermatozoa in both sperm preparations. In the study of Friedler et al. (1997) up to six fine needle punctures were performed in one testicle and sperm retrieval by open biopsy was applied to the other testicle. In our protocol, up to 15 punctures are performed in each testicle, increasing the amount of testicular aspirates up to 5-fold. Furthermore, sperm preparation in the study of Friedler et al. (1997) includes treatment of the cell suspension by erythrocyte-lysing buffer (Nagy et al., 1997), Percoll gradient separation in some cases and two additional washing steps. This complex procedure may cause the loss of spermatozoa during the various steps. Our sperm preparation protocol for testicular spermatozoa includes only one step of concentration by high speed (1800 g) centrifugation. When comparing these two methods, one should also take into account the recent observations of Schlegel and Su (1997). These studies demonstrated that following TESE, 82% of patients had intratesticular abnormalities present on ultrasound, suggestive of persistent haematoma and/or inflammation for as long as 3 months following the procedure. The majority of these lesions were transient and appeared to resolve by 6 months after the operation. However, permanent devascularization of the testis and extensive fibrosis were shown to occur following TESE procedures with multiple biopsies. In contrast, as in our study described above in which fine needle aspiration was performed, Watkins et al. (1997) found no major complications. Although the fine needle aspiration approach seems to be less invasive and to cause fewer complications, more studies on the long-term effects of the two techniques on testicular tissue is warranted before a consensus can be reached on the preferred method for the treatment of these patients. Such a prospective study is currently being performed in our centre. Conclusions In the last few years, ICSI has provided a remarkably effective solution for severe male factor infertility. Clinical interest in the fertilizing ability of epididymal spermatozoa developed originally from cases of obstructive azoospermia that could not be corrected surgically. It is now well established that ICSI 56

11 Treatment of azoospermia using epididymal as well as testicular spermatozoa in men with various causes of obstruction provides high fertilization and pregnancy rates with normal deliveries. MESA has been the main method of sperm retrieval in those patients, but other sperm retrieval techniques (PESA, TESA, TEFNA) were also introduced and proved to be equally successful. Recovery of testicular spermatozoa is now performed also in azoospermic patients with severely deficient spermatogenesis. Various studies have focused on the remarkable potential of retrieving viable spermatozoa from the testis using different retrieval methods. Although the ultimate choice of sperm retrieval method from the testis will depend on a number of factors including the clinical diagnosis, patient's preference and the availability of the necessary surgical skills, one of the most important parameters will be the physiological consequences of these techniques on testicular function. It is also very important to determine the prognostic factors which may predict a successful recovery procedure. For the time being, no strong preoperative predictors for successful sperm recovery except for testicular histology have been established (Silber et al., 1997b; Tournaye et al., 1997). It now appears that most types of male infertility, even such extreme cases as maturation arrest, Sertoli cell only, post-chemotherapy azoospermia and Klinefelter syndrome, can be successfully treated. Ironically, female age and ovarian reserve, rather then the source, the quantity or the quality of spermatozoa, are the major determinants of the success of assisted reproduction treatment in these patients (Silber et al, 1997a). Finally, although no direct evidence exists whether assisted reproductive techniques such as ICSI using testicular spermatozoa may compromise the reproductive health of the progeny by transmission of genetic deletions associated with azoospermia (Silber et al., 1995a), patients undergoing these procedures should be properly counselled. References Abuzeid, M.I., Chan, Y.M., Sasy, M.A. et al. (1995) Fertilization and pregnancy achieved by intracytoplasmic injection of sperm retrieved from testicular biopsies. Fertil. Steril., 64, Bourne, H., Watkins, W., Speirs, A. and Baker, H.W.G. (1995) Pregnancies after intracytoplasmic injection of sperm collected by fine needle biopsy of the testis. Fertil. Steril., 64, 433^36. Cha, K.Y., Oum, K.B. and Kim, H.J. (1997) Approaches for obtaining sperm in patients with male factor infertility. Fertil. Steril., 67, Craft, I. and Tsirigotis, M. (1995) Simplified recovery, preparation and cryopreservation of testicular spermatozoa. Hum. Reprod., 10, Craft, I., Bennett, V. and Nicholson, N. (1993) Fertilising ability of testicular spermatozoa. Lancet, 342, 864. Craft, I.L., Khalifa, Y., Boulos, A. et al. (1995a) Factors influencing the outcome of in-vitro fertilization with percutaneous aspirated epididymal spermatozoa and intracytoplasmic sperm injection in azoospermic men. Hum. Reprod., 10, Craft, I., Tsirigotis, M., Bennett, V. et al. (1995b) Percutaneous epididymal sperm aspiration and intracytoplasmic sperm injection in the management of infertility due to obstructive azoospermia. Fertil. Steril, 63,

12 A.Safran et al. Devroey, P., Liu, J., Nagy, Z. et al. (1994) Normal fertilization of human oocytes after testicular sperm extraction and intracytoplasmic sperm injection. Fertil. SteriL, 62, Devroey, P., Liu, J., Nagy, Z. et al. (1995a) Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in non-obsrtuctive azoospermia. Hum. Reprod., 10, Devroey, P., Silber, S., Nagy, Z. et al. (1995b) Ongoing pregnancies and birth after intracytoplasmic sperm injection with frozen-thawed epididymal spermatozoa. Hum. Reprod., 10, Devroey, P., Nagy, P., Tournaye, H. et al. (1996) Outcome of intracytoplasmic sperm injection with testicular spermatozoa in obstructive and non-obstructive azoospermia. Hum. Reprod., 11, Friedler, S., Raziel, A., Strassburger, D. et al. (1997) 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., 12, Gil-Salom, M., Remohi, J., Miguez, Y. et al. (1995) Pregnancy in an azoospermic patient with markedly elevated serum follicle stimulating hormone levels. Fertil. SteriL, 64, Gil-Salom, M., Romero, J., Minguez, Y. et al. (1996) Pregnancies after intracytoplasmic sperm injection with cryopreserved testicular spermatozoa. Hum. Reprod., 11, Goldstein, M., Young, G.P.H. and Einer-Jensen, N. (1983) Testicular artery damage due to infiltration with a fine gauge needle: experimental evidence suggesting that blind cord block should be abandoned. Snrg. Forum, 24, Hirsh, A., Mills, C, Bekir, J. etal. (1994) Factors influencing the outcome of in-vitro fertilization with epididymal spermatozoa in irreversible obstructive azoospermia. Hum. Reprod., 9, Hovatta, O., Moilanen, J., Von Smitten, K. and Reima, I. (1995) Testicular needle biopsy, open biopsy, epididymal aspiration and intracytoplasmic sperm injection in obstructive azoospermia. Hum. Reprod., 10, Kahraman, S., Ozgur, S., Alatas, C. et al. (1996a) Fertility with testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermic men. Hum. Reprod., 11, Kahraman, S., Ozgur, S., Alatas, C. et al. (1996b) High implantation and pregnancy rates with testicular sperm extraction and intracytoplasmic sperm injection in obstructive and nonobstructive azoospermia. Hum. Reprod., 11, Levin, H. S. (1979) Testicular biopsy in the study of male infertility. Its current usefulness, histologic techniques and prospects for the future. Hum. Pathol, 10, Lewin, A., Weiss, D.B., Friedler, S. et al. (1996) Delivery following intracytoplasmic injection of mature sperm cells recovered by testicular fine needle aspiration in a case of hypergonadotropic azoospermia due to maturation arrest. Hum. Reprod., 11, Mansour, R.T., Aboulghar, M.A., Serour, G.I. et al. (1996) Intracytoplasmic sperm injection using microsurgically retrieved epididymal and testicular sperm. Fertil. SteriL, 65, Mansour, R.T., Kamal, A., Fahmy, I. et al. (1997) Intracytoplasmic sperm injection in obstructive and non-obstructive azoospermia. Hum. Reprod., 12, Nagy, Z., Liu, J., Cecile, J. et al. (1995) Using ejaculated, fresh and frozen-thawed epididymal and testicular spermatozoa gives rise to comparable results after intracytoplasmic sperm injection. Fertil. SteriL, 63, Nagy, Z.P., Verheyen,G., Tournaye, H. et al. (1997) An improved treatment procedure for testicular biopsy specimens offers more efficient sperm recovery: case series. Fertil. SteriL, 68, Oates, R.D., Lobel, S.M., Harris, D.H. et al. (1996) Efficacy of intracytoplasmic sperm injection using intentionally cryopreserved epididymal spermatozoa. Hum. Reprod., 11, Oates, R.D., Mulhall, J., Burgess, C. et al. (1997) Fertilization and pregnancy using intentionally cryopreseved testicular tissue as the sperm source for intracytoplasmic sperm injection in 10 men with non-obstructive azoospermia. Hum. Reprod., 12, Palermo, G., Joris, H., Devroey, P. and Van Stierteghem, A.C. (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet, 340, Podsiadly, B.T., Woolcott, R.J., Stanger, J.D. and Stevenson, K. (1996) Pregnancy resulting from intracytoplasmic injection of cryopreserved spermatozoa recovered from testicular biopsy. Hum. Reprod., 11, Reubinoff, B.E., Abeliovich, D., Werner, M. et al. (1998) Ongoing pregnancy in non-mosaic 58

13 Treatment of azoospermia Klinefelter's syndrome after testicular fine needle aspiration, intracytoplasmic sperm injection and preimplantation genetic diagnosis. Hum. Reprod. 13, Schlegel, P. and Su, L.M. (1997) Physiological consequences of testicular sperm extraction. Hum. Reprod., 12, Schoysman, R., Vanderzwalmen, P., Nijs, M. et al. (1993) Pregnancy after fertilization with human testicular spermatozoa. Lancet, 342, Shrivastav, P., Nadkarni, P., Wensvoort, S. and Craft, I. (1994) Percutaneous epididymal sperm aspiration for obstructive azoospermia. Hum. Reprod., 11, Silber, S.J., Ord, T., Balmaceda, J. et al. (1990) Congenital absence of the vas deferens: the fertilizing capacity of human epididymal sperm. N. Eng. J. Med., 323, Silber, S., Nagy, Z., Liu, J. et al. (1994) Conventional in-vitro fertilization versus intracytoplasmic sperm injection for patients requiring microsurgical sperm aspiration. Hum. Reprod., 9, Silber, S.J., Nagy, Z., Liu, J. et al. (1995a) The use of epididymal and testicular spermatozoa for intracytoplasmic sperm injection: The genetic implications for male infertility. Hum. Reprod., 10, Silber, S.J., Van Steirteghem, A.C. and Devroey, P. (1995b) Sertoli cell only revisited. Hum. Reprod., 10, Silber, S.J., Van Steirteghem, A.C, Liu, J. et al. (1995c) High fertilization and pregnancy rate after intracytoplasmic sperm injection with spermatozoa obtained from testicle biopsy. Hum. Reprod., 10, Silber, S.J., Van Steirteghem, A., Nagy, Z. et al. (1996) Normal pregnancies resulting from testicular sperm extraction and intracytoplasmic sperm injection for azoospermia due to maturation arrest. Fertil. Steril., 66, Silber, S., Nagy, Z., Devroey, P. et al. (1997a) The effect of female age and ovarian reserve on pregnancy rate in male infertility: treatment of azoospermia with sperm retrieval and intracytoplasmic sperm injection. Hum. Reprod., 12, Silber, S., Nagy, Z., Devroey, P. et al. (1997b) Distribution of spermatogenesis in the testicles of azoospermic men: the presence or absence of spermatids in the testes of men with germinal failure. Hum. Reprod., 12, Staessen, C, Coonen, E., Van Assche, E. et al. (1996) Preimplantation diagnosis for X and Y normality in embryos from three Klinefelter patients. Hum. Reprod., 11, Staessen, C, Van Assche, E., Joris, H. et al. (1997) Preimplantation diagnosis by fluorescent insitu hybridisation in embryos from 47,XXY or 47,XXX patients. J. Assist. Reprod. Genet., 14, 468 (abstract). Temple Smith, P.D., Southwick, G.J., Yates, C.A. et al. (1985) Human pregnancy by in-vitro fertilisation (IVF) using sperm aspirated from the epididymis. /. In Vitro Fertil. Embryo Transfer, 2, Tournaye, H., Devroey, P., Liu, J. et al. (1994) Microsurgical epididymal sperm aspiration and intracytoplasmic sperm injection: a new effective approach to infertility as a result of congenital bilateral absence of the vas deferens. Fertil. Steril., 61, Tournaye, H., Camus, M., Goossens, A. et al. (1995) Recent concepts in the management of infertility because of non-obstructive azoospermia. Hum. Reprod., 10 (Suppl. 1), Tournaye, H., Liu, J., Camus, M. et al. (1996a) Correlation between testicular histology and outcome after intracytoplasmic sperm injection using testicular spermatozoa. Hum. Reprod., 11, Tournaye, H., Staessen, C, Liebaers, I. et al. (1996b) Testicular sperm recovery in nine 47,XXY Klinefelter patients. Hum. Reprod., 11, Tournaye, H., Verheyen, G., Nagy, P. et al. (1997) Are there any predictive factors for successful testicular sperm recovery in azoospermic patients? Hum. Reprod., 12, Tsirigotis, M. and Craft, I. (1995) Sperm retrieval methods and ICSI for obstructive azoospermia. Hum. Reprod., 10, Tsirigotis, M. and Craft, I. (1996) Micro-epididymal sperm aspiration or percutaneous epididymal sperm aspiration? The dilemma. Hum. Reprod., 11, Tsirigotis, M., Pelekanos, M., Beski, S. et al. (1996) Cumulative experience of percutaneous 59

14 A.Safran et al. epididymal sperm aspiration (PESA) with intracytoplasmic sperm injection. J. Assist. Reprod. Genet., 13, Van Steirteghem, A.C., Nagy, Z., Joris, H. et al. (1993) High fertilization and implantation rates after intracytoplasmic sperm injection. Hum. Reprod., 8, Watkins, W., Nieto, E, Bourne, H. et al. (1997) Testicular and epididymal sperm in a microinjection program: methods of retrieval and results. Fertil. Steril., 67, Yemini, M., Vanderzwalmen, P., Mukaida, T. et al. (1995) Intracytoplasmic sperm injection, fertilization and embryo transfer after retrieval of spermatozoa by testicular biopsy from an azoospermic male with testicular tubular atrophy. Fertil. Steril., 63,