Assisted reproduction for the treatment of azoospermia

Assisted reproduction for the treatment ofazoospermia

Anat Safran1, Benjamin E.Reubinoff, Anat Porat-Katz,Joseph G.Schenker and Aby Lewin

IVF Unit, Department of Obstetrics and Gynecology, Hadassah HebrewUniversity 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 obstruc-tions in the genital tract or by testicular failure. Microsurgical techniquesare available for the correction of some of these obstructions but no effectivetreatment is available for testicular failure. In recent years, methods havebeen developed for direct surgical sperm sampling from either the epididymisor the testis to be used by intracytoplasmic sperm injection. The mainapproach proven to be effective for the retrieval of spermatozoa fromthe epididymis in patients with obstructive azoospermia is microsurgicalepididymal sperm aspiration, although recently the retrieval of spermatozoaby fine needle aspiration was shown to be equally effective. Recovery ofspermatozoa is also now performed in patients with severely deficientspermatogenesis using testicular open biopsy as well as aspiration by fineneedle. The ultimate choice of sperm retrieval method in these patientswill depend not only on sperm availability, but also on the physiologicalconsequences of the different techiques on testicular function. This articlesummarizes the recent advances achieved in the treatment of azoospermicpatients 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 avariety of endocrine, genetic and immunological aetiologies, as well as sexualmalfunction, varicocele and genital infections. The most severe form of maleinfertility is azoospermia, a term originating from the Greek words azoos (lifeless)and sperma (seed). Thus, azoospermia defines the condition of absence ofspermatozoa in the semen and is due to either obstruction in the genital tract ortesticular failure. In obstructive azoospermia the process of spermatogenesis isnot impaired. The most common causes of obstruction are epididymovasalocclusions 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

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available for surgical correction of these obstructions. Epididymovasostomy, themain procedure performed for the correction of post-infection obstruction, wasshown to provide 32-47% success in achieving patency. Vasovasostomy isperformed mainly for reversal of vasectomy and patency was reported to beachieved in 30-88% of patients with a pregnancy rate of 18-53%. The majoradvantage of successful microsurgical intervention is the possibility of achievingmultiple pregnancies following a single operation. Thus, when the operativeskills are available, microsurgery is offered to patients for reversal of vasectomyand for selected cases with epididymovasal occlusions. With the new advancesin assisted reproductive techniques, patients with poor surgical prognosis cannow be referred to surgical procedures of sperm aspiration to be used for in-vitrofertilization (IVF).

Non-obstructive azoospermia is defined as a condition in which there is animpairment of various degrees in the process of spermatogenesis. For patientswith 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 orhypothalamic deficiency and treatment includes hormonal supplementation forthe duration of time required for resumption of spermatogenesis. Thus, the onlyapproach for treatment for non-hypogonadotrophic, non-obstructive, azoospermicpatients is a surgical intratesticular attempt for the retrieval of spermatozoa. Thecorrect differential diagnosis of obstructive and non-obstructive azoospermia isimportant 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 preoperativeclinical features and testicular histology. Furthermore, recent surgical attemptsto recover spermatozoa in cases of apparent testicular failure have provedsuccessful in many cases, thus further reducing the prognostic value of theclinical parameters. Final prognosis of sperm availability in azoospermic patientscan therefore be achieved only with the combination of the clinical parametersand direct surgical sperm sampling.

This article summarizes the advances achieved during recent years in thetreatment of patients with both obstructive as well as non-obstructive azoospermiausing assisted reproductive techniques.

Sperm retrieval methods for the treatment of obstructive azoospermia

The first azoospermic patients to be treated by assisted reproduction techniqueswere those with obstructive azoospermia. Microsurgical epididymal spermaspiration (MESA) combined with IVF has led to the achievement of viablepregnancies (Temple Smith et al., 1985; Silber et al., 1990) but was found tooffer 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

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Treatment of azoospermia

Table I. Clinical features related to obstructive and non-obstructive azoospermia

Obstructive (excretory) Non-obstructive

Patient's history

EpididymitisHernia repairProstatic or bladder surgeryCystic fibrosisVasectomyNeurogenic disorders:

Retrograde ejaculationImpaired contractility of vas and vesicles in

diabetes following lymph node dissection(testicular tumour)

Serum follicle stimulating hormone

Usually normal

Physical examination

Normal testicular sizeScrotal testicular locationPossible absence of vasPossible induration of epididymis

Cryptorchidisma

Mumps orchitisTorsionTraumaChemotherapyIrradiation

Elevated/normalLow in hypogonadotropic hypogonadism

Small/normal testicular sizeScrotal testicular location/cryptorchidismGynaecomastia in Klinefelter

Seminal markers

Normal/absent/low fructose (seminal vesicles)13 Normal fructose and carnitineand low carnitine (epididymis)

Testicular biopsy

Normal spermatogenesis Germinal hypoplasia (hypospermatogenesis)Spermatogenic arrest (maturation arrest)Germinal aplasia (Sertoli cells only)Tubular hyalinizationTesticular fibrosis

aCryptorchidism may cause irreversible testicular damage and azoospermia, depending on thetiming and efficiency of medical (human chorionic gonadotrophin) and surgical treatment.bFructose is absent or low in some patients with congenital absence of the vas deferens and insome patients with obstructions of the ejaculatory ducts.

patients. The efficacy of combining MESA with ICSI for the treatment ofinfertility due to CBAVD was analysed (Tournaye et al., 1994), demonstratinghigh fertilization (58%) and pregnancy (35.7%) rates even with the use of grosslyimpaired epididymal spermatozoa. MESA is generally performed under generalanaesthesia. After unilateral hemiscrototomy, the epididymis is explored usingan operating microscope and carefully dissected to open several epididymaltubules by microscissors. The epididymal fluid is aspirated using a glass pipetteand transferred into a conical test tube. Sperm preparation usually includesseparation on a two-layer Percoll gradient. A randomized comparative studyaimed to determine whether ICSI could achieve better results than conventionalIVF with microsurgical aspiration of spermatozoa in patients with CBAVD or

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irreparable obstructions (Silber et al., 1994). Results showed high fertilizationrates and the development of normal embryos in 82% of MESA/ICSI cycles ascompared to 19% with conventional IVF. Overall fertilization and pregnancyrates were significantly higher using ICSI, being 45% and 47% as compared to6.9% and 4.5% for conventional IVF, respectively. It was thus suggested thatICSI may be mandated for all future MESA patients. It has been suggested thatthe aetiology of obstruction may affect treatment results. Indeed, in a recentstudy by Mansour et al. (1997), a significant difference in pregnancy rate wasobserved 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 obstructedepididymis was the inversion of the expected pattern of motility, usually best inthe most proximal region (Silber et al., 1990). Furthermore, a remarkably highpregnancy wastage was reported by Tournaye et al. (1994), who retrievedspermatozoa mainly from the cauda. The observation that sperm cells from amore proximal region of the epididymis provide high fertilization rates by ICSImay indicate that although passage through the epididymis is required for normalfertilization, it is not a prerequisite for a sperm cell to gain its intrinsic abilityto fertilize the egg and result in normal embryo development once penetrationis accomplished by ICSI. The combination of MESA/ICSI also enabled thefreezing of epididymal spermatozoa to be used successfully in additional ICSIcycles (Devroey et al., 1995b) avoiding repeated scrotal surgery. In a recentstudy by Oates et al. (1996), frozen-thawed epididymal spermatozoa were shownto lead to equivalent fertilization and pregnancy rates as compared with freshepididymal spermatozoa. Based on these results, a new approach was suggestedin which sperm and oocyte harvesting need not be performed simultaneously.This scheme may ease the burden of partner scheduling and assure the availabilityof spermatozoa prior to ovulation induction, thus avoiding unnecessary ovarianstimulation and oocyte retrieval.

In some patients requiring MESA for obstructive azoospermia, often nospermatozoa can be retrieved from the epididymis due to extensive scarring fromprevious corrective microsurgery or MESA procedures, or due to hypoplasia. Insome cases the epididymis on both sides is completely absent. In these patients,the only source of spermatozoa is within the testicles. The fertilizing ability oftesticular spermatozoa obtained by open testicular biopsy was demonstrated forthe first time in a patient in which epididymal aspiration failed (Craft et al,1993). The first pregnancy following the use of testicular spermatozoa obtainedby testicular biopsy in a patient in which epididymal sperm retrieval failed(Schoysman et al., 1993), offered a new opportunity for patients with inoperableexcretory occlusion. In a small series described by Devroey et al. (1994), theassociation of testicular sperm extraction (TESE) and ICSI yielded a highfertilization rate in males with absence of the epididymis. In a larger series ofobstructive patients, a study was undertaken to compare the results obtainedusing epididymal spermatozoa to those obtained using testicular spermatozoawhen epididymal aspiration failed or when the epididymis was absent or severely

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scarred (Silber et al, 1995c). The objective of this study was to determinewhether ICSI could salvage the most severe MESA cases where there wereabsolutely no epididymal spermatozoa available, by resorting to TESE. Theprocedure is usually performed under general anaesthesia. An incision is madein the scrotal skin and carried through the peritoneal tunica vaginalis. A smallincision is then made in the tunica albuginea and the small portion of extrudingtesticular tissue is excised and minced in HEPES-buffered medium. The effluentis centrifuged and the pellet resuspended in medium and kept in the incubatoruntil 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 bothepididymal and testicular origin yield similar fertilization, cleavage and ongoingpregnancy rates using ICSI, although the main disadvantage of testicularspermatozoa was the low number of sperm cells retrieved and their poor motility,making cryopreservation of these cells more problematic than with epididymalspermatozoa (Silber et al., 1995c). In another study (Nagy et al., 1995), the poorparameters of testicular spermatozoa were compared to epididymal and ejaculatedspermatozoa. The mean total free sperm count of testicular spermatozoa was0.54X 106 as compared to 46.2X 106 and 17.8X 106 for epididymal and ejaculatedspermatozoa, respectively. Testicular sperm always showed only a slow andsluggish local motility, but the number of motile spermatozoa slightly increasedafter 1-2 h of in-vitro incubation. It was thus suggested that in cases of obstructiveazoospermia, the MESA/ICSI technique should be attempted first, allowingmultiple aliquots to be frozen and used in subsequent cycles. In cases whereepididymal spermatozoa are not retrievable, testicular biopsy could be a finalresort that is also very effective, providing most patients with embryos fortransfer (Abuzeid et al., 1995; Mansour et al., 1996). Despite the lower numberof sperm cells retrieved from the testis and their extremely poor motility, thefertilizing ability of these cells was shown to be retained following cryopreserv-ation, as pregnancies were achieved following the injection of cryopreservedspermatozoa recovered from testicular biopsies in cases of obstructive azoosper-mia (Gil-Salom et al, 1996; Podsiadly et al, 1996).

Consequently, successful sperm aspiration for the treatment of obstructiveazoospermia was reported using a less invasive approach of sperm retrieval fromthe epididymis by fine needle aspiration. In the first study (Shrivastav et al,1994), percutaneous epididymal sperm aspiration (PESA) was applied for thetreatment of seven patients with obstructive azoospermia. The procedure isperformed either under general or local anaesthesia and the aspiration apparatusincludes a 25-gauge butterfly needle connected to a 1 ml tuberculin syringe. Theneedle is directly inserted into the epididymis, suction is applied and the aspiratesare washed into a sterile tube. Spermatozoa are usually prepared by a discontinuousPercoll gradient. In the study by Shrivastav et al (1994), high sperm countswere achieved in all cases (0.8-200XlO6/ml) and three pregnancies wereestablished. In a larger series (Tsirigotis et al, 1996), a high recovery rate(85.5%) of spermatozoa from the epididymis using the percutaneous aspirationapproach, as well as high fertilization (52.6%) and pregnancy (33.3%) rates were

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reported. These results were comparable to those achieved applying MESA forthe treatment of obstructive azoospermic patients (Silber et al., 1994). The qualityof spermatozoa retrieved by PESA varied widely and it was suggested that ICSIshould be used to maximize fertilization rates (Craft et al., 1995a). In light ofthese studies, it was concluded that the PESA technique is simple, efficient anddoes not require specific microsurgical skills. It was also suggested to beassociated with fewer complications than an open microsurgical operation (Craftet al., 1995a,b). A similar approach of percutaneous sperm aspiration from thetestis (TESA; Craft and Tsirigotis, 1995) evolved from the PESA procedure. Itwas suggested that TESA be applied to men with active spermatogenesis, withor without an obstruction, in situations where spermatozoa are not recoveredfollowing MESA/PESA operations and for some non-obstructive cases ofazoospermia. The advantages of the percutaneous approach raised in relation toPESA were relevant for the TESA technique as well, especially in view of thefact 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, wehave also adopted the percutaneous approach combining testicular and epididymalfine needle aspiration (TEFNA) for the treatment of patients with obstructiveazoospermia (A.Safran et al., unpublished data). The technique is performed ina similar manner to previous publications (Shrivastav et al., 1994; Craft et al.,1995b), with some modifications. The aspiration set-up includes a 23-gaugebutterfly needle connected to a 20 ml syringe installed in an aspiration handlefor the creation of steady suction. The needle is introduced into the caput of theepididymis and suction is established by using the aspiration handle device.Following each aspiration, when epididymal fluid ceases to accumulate in thetube, the needle is withdrawn and the aspirate is washed into a 4-well platecontaining HEPES-buffered medium. Sperm is prepared using a two-layer Percollgradient. Eleven patients underwent 12 treatment cycles. In 11 cycles, spermcells were easily recovered from the caput region of the epididymis and onlyone or two punctures were needed. For one patient, spermatozoa could not beretrieved from the epididymis as a result of severe scarring following previousMESA operations. Spermatozoa were recovered by fine needle aspiration fromthe testes using the same set-up. Mature sperm cells were available in all 12cycles and five clinical pregnancies (41%) were achieved. In 11 cycles, sparespermatozoa were cryopreserved. No complications were noted in any of thesepatients. We thus concluded that by employing this relatively simple, inexpensiveand well-tolerated technique, sperm recovery can be predicted for almost allpatients with obstructive azoospermia. If spermatozoa are not retrieved from theepididymis, TEFNA can immediately be performed using exactly the same set-up.

Concern has been expressed as to whether blind needle puncture of theepididymis or the testis can cause damage and post-operative complications suchas 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 tothe testicular vasculature in the spermatic cord. Such risks are significantly

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reduced in humans because of the use of fine needles, the size of the testis andthe fact that puncture of both the epididymis and the testis is performed awayfrom the main vascular supply (Craft and Tsirigotis, 1995). Indeed, post-operativecomplications of patients undergoing these procedures were minimal, with fewpatients complaining of short-term mild pain. Despite the simplicity of the TESAmethod, it was still suggested that PESA should be the primary aspirationtechnique for men with obstructions, because of the ability to recover largernumbers of motile spermatozoa from the epididymis, some of which can becryopreserved for future use (Tsirigotis and Craft, 1996).

There seem to be differences in the number of sperm cells obtained by thetwo sperm retrieval techniques in obstructive azoospermic patients, with therecovery of fewer spermatozoa using the percutaneous approach. The number ofspermatozoa retrieved by TEFNA is in the order of hundreds of thousands to afew millions. Since only a few dozen motile spermatozoa are required pertreatment cycle, the number of spermatozoa retrieved using TEFNA enabledtheir use in all treatment cycles as well as their cryopreservation for severalfuture attempts in most cases.

A few other reports have been published in which attempts were made toavoid the need of large tissue sampling for the treatment of obstructiveazoospermia. A modified percutaneous sperm aspiration technique was describedby Cha et al. (1997). A small incision is made in the scrotum, creating a directview of the epididymis. Sperm cells are aspirated using a 24-gauge angio-needleand sperm preparation is performed as in the MESA approach. High fertilization(77.3%) and pregnancy (43.5%) rates were achieved using this technique and itwas suggested that making this small opening may reduce the trauma caused byblind puncture of the epididymis. Hovatta et al. (1995) described an alternativetechnique of testicular sperm retrieval by using a biopsy gun needle. Using thisapproach, small pieces of testicular tissue could be sampled from a wider areaof the testis and spermatozoa were obtained in 82% of cycles. Bourne et al.(1995) reported successful aspiration of testicular spermatozoa by a 20-gaugeMenghini biopsy needle. Therefore, it seems that simple percutaneous aspirationor needle biopsy (Watkins et al., 1997) offers a high sperm recovery rate whilebeing easy to perform, well tolerated by the patients and cost effective.

Sperm retrieval methods for the treatment of non-obstructiveazoospermia

In non-obstructive azoospermia, the process of spermatogenesis is impaired, theepididymis is usually devoid of spermatozoa, and in some of these patients onlya few foci with complete spermatogenesis may exist in the testicles (Silber etah, 1995b). Initially, few case reports were published in which successful spermrecovery by TESE was achieved in patients with testicular failure. Yemini et al.(1995) reported fertilization following the injection of few spermatozoa obtainedby open testicular biopsy in an azoospermic patient with almost complete

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spermatogenic arrest and testicular tubular atrophy. In another study, an azoos-permic patient with small testes, high levels of serum follicle stimulatinghormone (FSH) and testicular histology of partial germinal aplasia with focalspermatogenesis was treated by TESE (Gil-Salom et al, 1995). A few spermatozoawith 'twitching' motility were recovered, leading to a high fertilization rate(73%) and a pregnancy. In this case report it was demonstrated for the first timethat patients with almost complete spermatogenic failure and markedly elevatedFSH levels may be successfully treated by TESE combined with ICSI. Concomit-antly with these studies, the efficacy of ICSI with TESE in a series of 15 patientswith non-obstructive azoospermia was analysed (Devroey et al, 1995a). In thisstudy, the inclusion of patients suffering from non-obstructive azoospermia wasmainly based on high FSH concentrations and small testicular size. Consequently,the histology of these patients revealed severe spermatogenic defects in mostcases. A high sperm recovery rate (86%) was reported, and the fertilization(47.8%) and pregnancy (25%) rates achieved were remarkably similar to thoseobtained using testicular spermatozoa from patients with normal spermatogenesis(Devroey et al., 1994). Further experience of several groups with TESE andICSI for the treatment of non-obstructive azoospermic patients has been recentlypublished (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 fewimportant conclusions could be drawn. It was suggested that an attempt of spermrecovery should be offered to all azoospermic patients, irrespective of serumFSH levels, testicular size or medical history (Tournaye et al, 1995; Mansour etal, 1997). When the outcome of treatment was analysed in relation to testicularhistology, no correlation was found between histological diagnosis and spermrecovery in the wet preparation (Tournaye et al, 1996a). This pointed out theshortcoming of a single testicular specimen in making the correct histologicaldiagnosis and could be explained by the focal nature of spermatogenesis inthese patients. It was also demonstrated that the normal fertilization rate wassignificantly reduced in oocytes injected with spermatozoa deriving from menwith germ-cell aplasia and maturation arrest (Tournaye et al, 1996b). However,once normal fertilization was achieved, embryonic development was comparablewith other infertility aetiologies. The fertilizing ability of sperm cells recoveredfrom cryopreserved testicular tissue obtained by TESE, which led to theachievement of viable pregnancies from non-obstructive azoospermic patients,was recently demonstrated (Oates et al, 1997). Because of the difficulty inpredicting the availability of spermatozoa in these patients, it was suggested inthis study that sperm recovery should be performed prior to initiation of an ICSIcycle. If spermatozoa are found, the tissue can be cryopreserved and become the'sperm source' for multiple ICSI cycles. This approach could prevent surgicalintervention in the female partner when sperm recovery fails.

Histological analysis of patients with non-obstructive azoospermia has demon-strated that if the entire testis is carefully sampled, occasional foci of completespermatogenesis 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

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(1997b), the distribution of active areas of spermatogenesis in cases of testicularfailure was suggested to be evenly scattered throughout the testicle, rather thanin a few patchy areas. Nevertheless, the concept of spermatogenesis beingconfined to patchy areas in non-obstructive azoospermic patients was the basisfor a study that we undertook in order to evaluate the feasibility of obtainingspermatozoa by multiple sampling of the entire testis by TEFNA in an azoospermicpatient with maturation arrest (Lewin et al., 1996). Eight sperm cells withapparently normal morphology and signs of motility were found and one oocytewas normally fertilized out of four mature oocytes injected. A single embryowas transferred and a pregnancy and delivery of a healthy boy were achieved.We postulate that unlike a single testicular biopsy, in cases of testicular failurethe TEFNA approach may enable the surgeon to reach multiple areas and thusincrease the chance of 'hitting' a rare site of active spermatogenesis. Based onthis hypothesis, we initiated a prospective study in which epididymal andtesticular fine needle aspiration was applied to all non-obstructive azoospermicpatients (Lewin, A., Safran, A., Reubinoff, B.P. et al. unpublished data, 1998).Confirmation of the non-obstructive nature of azoospermia, according to theclassification by Levin (1979), was made from the histological report of eitherthe diagnostic preoperative biopsy, or from tissue sampling during the spermretrieval procedure. Pseudo azoospermia was excluded from this group, as allmen submitted ejaculate samples on two occasions prior to surgery and thesesamples underwent extensive examination after high velocity centrifugation andwere all found to be devoid of any spermatozoa. Of 85 patients, spermatozoacould 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 asonly three patients suffered pain necessitating treatment with oral analgesia. Insummary, a high sperm recovery rate could be achieved applying TEFNA forthe treatment of non-obstructive azoospermic patients and, once spermatozoawere retrieved, the outcome was comparable to that achieved by other spermretrieval methods. With regard to the dispute concerning the distribution ofspermatogenic sites in the testicles of non-obstructive azoospermic patients, ourexperience supports the hypothesis of focal spermatogenesis because, in manycases during the first search of the wet preparation, spermatozoa could be detectedin one out of a few tens of aspiration samples. Spermatozoa could be recoveredeven in four of six cases of Klinefelter syndrome and a delivery followingpreimplantation diagnosis and the transfer of a normal 46XY embryo wasachieved (Reubinoff et al., 1998). Sperm recovery from non-mosaic 47XXYKlinefelter patients using TESE was also reported (Tournaye et al., 1996b) aswell as the constitution of sex chromosomes in the embryos obtained from threeof these patients (Staessen et al., 1996). The conception achieved in our centreusing TEFNA (Reubinoff et al., 1998), is among the first few pregnancies(Staessen et al., 1997) obtained so far using spermatozoa aspirated fromazoospermic non-mosaic Klinefelter individuals. We thus suggest that TEFNAmay be considered as an alternative approach to TESE for the treatment of non-obstructive azoospermic patients.

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Recently, the efficiency of testicular sperm retrieval by TEFNA was comparedwith open biopsy and TESE "in 37 rigorously selected patients with non-obstructive azoospermia (Friedler et al., 1997). All patients underwent bothretrieval techniques, thus each patient served as his own control. Whereas byTEFNA, spermatozoa enabling the performance of ICSI were found in fourpatients out of 37 (11%), open biopsy and TESE yielded spermatozoa in 16cases (43%). Several explanations could be given for the discrepancy in the rateof sperm retrieval achieved in this study as compared to ours, applying TEFNAfor patients with proven testicular failure. In our protocol, the search forspermatozoa, in most patients, required 5-8 h and in many cases only fewspermatozoa were found after several hours. If the two techniques, TEFNA andTESE, are performed sequentially on the same day, it is probably impossible toinvest the same amount of time for the search of spermatozoa in both spermpreparations. In the study of Friedler et al. (1997) up to six fine needle punctureswere performed in one testicle and sperm retrieval by open biopsy was appliedto the other testicle. In our protocol, up to 15 punctures are performed in eachtesticle, increasing the amount of testicular aspirates up to 5-fold. Furthermore,sperm preparation in the study of Friedler et al. (1997) includes treatment of thecell suspension by erythrocyte-lysing buffer (Nagy et al., 1997), Percoll gradientseparation in some cases and two additional washing steps. This complexprocedure may cause the loss of spermatozoa during the various steps. Oursperm preparation protocol for testicular spermatozoa includes only one step ofconcentration by high speed (1800 g) centrifugation. When comparing these twomethods, one should also take into account the recent observations of Schlegeland Su (1997). These studies demonstrated that following TESE, 82% of patientshad intratesticular abnormalities present on ultrasound, suggestive of persistenthaematoma 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 monthsafter the operation. However, permanent devascularization of the testis andextensive fibrosis were shown to occur following TESE procedures with multiplebiopsies. In contrast, as in our study described above in which fine needleaspiration was performed, Watkins et al. (1997) found no major complications.Although the fine needle aspiration approach seems to be less invasive and tocause fewer complications, more studies on the long-term effects of the twotechniques on testicular tissue is warranted before a consensus can be reachedon the preferred method for the treatment of these patients. Such a prospectivestudy is currently being performed in our centre.

Conclusions

In the last few years, ICSI has provided a remarkably effective solution forsevere male factor infertility. Clinical interest in the fertilizing ability ofepididymal spermatozoa developed originally from cases of obstructive azoosper-mia that could not be corrected surgically. It is now well established that ICSI

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using epididymal as well as testicular spermatozoa in men with various causesof obstruction provides high fertilization and pregnancy rates with normaldeliveries. MESA has been the main method of sperm retrieval in those patients,but other sperm retrieval techniques (PESA, TESA, TEFNA) were also introducedand proved to be equally successful.

Recovery of testicular spermatozoa is now performed also in azoospermicpatients with severely deficient spermatogenesis. Various studies have focusedon the remarkable potential of retrieving viable spermatozoa from the testis usingdifferent retrieval methods. Although the ultimate choice of sperm retrievalmethod from the testis will depend on a number of factors including the clinicaldiagnosis, patient's preference and the availability of the necessary surgical skills,one of the most important parameters will be the physiological consequences ofthese techniques on testicular function. It is also very important to determine theprognostic factors which may predict a successful recovery procedure. For thetime being, no strong preoperative predictors for successful sperm recoveryexcept 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 asmaturation arrest, Sertoli cell only, post-chemotherapy azoospermia and Kline-felter syndrome, can be successfully treated. Ironically, female age and ovarianreserve, rather then the source, the quantity or the quality of spermatozoa, arethe major determinants of the success of assisted reproduction treatment in thesepatients (Silber et al, 1997a).

Finally, although no direct evidence exists whether assisted reproductivetechniques such as ICSI using testicular spermatozoa may compromise thereproductive health of the progeny by transmission of genetic deletions associatedwith azoospermia (Silber et al., 1995a), patients undergoing these proceduresshould be properly counselled.

References

Abuzeid, M.I., Chan, Y.M., Sasy, M.A. et al. (1995) Fertilization and pregnancy achieved byintracytoplasmic injection of sperm retrieved from testicular biopsies. Fertil. Steril., 64, 644-646.

Bourne, H., Watkins, W., Speirs, A. and Baker, H.W.G. (1995) Pregnancies after intracytoplasmicinjection 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 malefactor infertility. Fertil. Steril., 67, 985-995.

Craft, I. and Tsirigotis, M. (1995) Simplified recovery, preparation and cryopreservation of testicularspermatozoa. Hum. Reprod., 10, 1623-1627.

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-vitrofertilization with percutaneous aspirated epididymal spermatozoa and intracytoplasmic sperminjection in azoospermic men. Hum. Reprod., 10, 1791-1794.

Craft, I., Tsirigotis, M., Bennett, V. et al. (1995b) Percutaneous epididymal sperm aspiration andintracytoplasmic sperm injection in the management of infertility due to obstructive azoospermia.Fertil. Steril, 63, 1038-1042.

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Devroey, P., Liu, J., Nagy, Z. et al. (1994) Normal fertilization of human oocytes after testicularsperm extraction and intracytoplasmic sperm injection. Fertil. SteriL, 62, 639-641.

Devroey, P., Liu, J., Nagy, Z. et al. (1995a) Pregnancies after testicular sperm extraction andintracytoplasmic sperm injection in non-obsrtuctive azoospermia. Hum. Reprod., 10, 1457-1460.

Devroey, P., Silber, S., Nagy, Z. et al. (1995b) Ongoing pregnancies and birth after intracytoplasmicsperm injection with frozen-thawed epididymal spermatozoa. Hum. Reprod., 10, 903-906.

Devroey, P., Nagy, P., Tournaye, H. et al. (1996) Outcome of intracytoplasmic sperm injectionwith testicular spermatozoa in obstructive and non-obstructive azoospermia. Hum. Reprod., 11,1015-1018.

Friedler, S., Raziel, A., Strassburger, D. et al. (1997) Testicular sperm retrieval by percutaneousfine needle sperm aspiration compared with testicular sperm extraction by open biopsy in menwith non-obstructive azoospermia. Hum. Reprod., 12, 1488-1493.

Gil-Salom, M., Remohi, J., Miguez, Y. et al. (1995) Pregnancy in an azoospermic patient withmarkedly elevated serum follicle stimulating hormone levels. Fertil. SteriL, 64, 1218-1220.

Gil-Salom, M., Romero, J., Minguez, Y. et al. (1996) Pregnancies after intracytoplasmic sperminjection with cryopreserved testicular spermatozoa. Hum. Reprod., 11, 1309-1313.

Goldstein, M., Young, G.P.H. and Einer-Jensen, N. (1983) Testicular artery damage due toinfiltration with a fine gauge needle: experimental evidence suggesting that blind cord blockshould be abandoned. Snrg. Forum, 24, 653-656.

Hirsh, A., Mills, C, Bekir, J. etal. (1994) Factors influencing the outcome of in-vitro fertilization withepididymal spermatozoa in irreversible obstructive azoospermia. Hum. Reprod., 9, 1710-1716.

Hovatta, O., Moilanen, J., Von Smitten, K. and Reima, I. (1995) Testicular needle biopsy, openbiopsy, epididymal aspiration and intracytoplasmic sperm injection in obstructive azoospermia.Hum. Reprod., 10, 2595-2599.

Kahraman, S., Ozgur, S., Alatas, C. et al. (1996a) Fertility with testicular sperm extraction andintracytoplasmic sperm injection in non-obstructive azoospermic men. Hum. Reprod., 11,756-760.

Kahraman, S., Ozgur, S., Alatas, C. et al. (1996b) High implantation and pregnancy rates withtesticular sperm extraction and intracytoplasmic sperm injection in obstructive and non-obstructive azoospermia. Hum. Reprod., 11, 673-676.

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, 569-584.

Lewin, A., Weiss, D.B., Friedler, S. et al. (1996) Delivery following intracytoplasmic injection ofmature sperm cells recovered by testicular fine needle aspiration in a case of hypergonadotropicazoospermia due to maturation arrest. Hum. Reprod., 11, 769-771.

Mansour, R.T., Aboulghar, M.A., Serour, G.I. et al. (1996) Intracytoplasmic sperm injection usingmicrosurgically retrieved epididymal and testicular sperm. Fertil. SteriL, 65, 566-572.

Mansour, R.T., Kamal, A., Fahmy, I. et al. (1997) Intracytoplasmic sperm injection in obstructiveand non-obstructive azoospermia. Hum. Reprod., 12, 1974-1979.

Nagy, Z., Liu, J., Cecile, J. et al. (1995) Using ejaculated, fresh and frozen-thawed epididymaland testicular spermatozoa gives rise to comparable results after intracytoplasmic sperm injection.Fertil. SteriL, 63, 808-815.

Nagy, Z.P., Verheyen,G., Tournaye, H. et al. (1997) An improved treatment procedure for testicularbiopsy specimens offers more efficient sperm recovery: case series. Fertil. SteriL, 68, 376-379.

Oates, R.D., Lobel, S.M., Harris, D.H. et al. (1996) Efficacy of intracytoplasmic sperm injectionusing intentionally cryopreserved epididymal spermatozoa. Hum. Reprod., 11, 133-138.

Oates, R.D., Mulhall, J., Burgess, C. et al. (1997) Fertilization and pregnancy using intentionallycryopreseved testicular tissue as the sperm source for intracytoplasmic sperm injection in 10men with non-obstructive azoospermia. Hum. Reprod., 12, 734-739.

Palermo, G., Joris, H., Devroey, P. and Van Stierteghem, A.C. (1992) Pregnancies afterintracytoplasmic injection of single spermatozoon into an oocyte. Lancet, 340, 17-18.

Podsiadly, B.T., Woolcott, R.J., Stanger, J.D. and Stevenson, K. (1996) Pregnancy resulting fromintracytoplasmic injection of cryopreserved spermatozoa recovered from testicular biopsy. Hum.Reprod., 11, 1306-1308.

Reubinoff, B.E., Abeliovich, D., Werner, M. et al. (1998) Ongoing pregnancy in non-mosaic58



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Klinefelter's syndrome after testicular fine needle aspiration, intracytoplasmic sperm injectionand preimplantation genetic diagnosis. Hum. Reprod. 13, 1887-1892.

Schlegel, P. and Su, L.M. (1997) Physiological consequences of testicular sperm extraction. Hum.Reprod., 12, 1688-1692.

Schoysman, R., Vanderzwalmen, P., Nijs, M. et al. (1993) Pregnancy after fertilization with humantesticular spermatozoa. Lancet, 342, 1237.

Shrivastav, P., Nadkarni, P., Wensvoort, S. and Craft, I. (1994) Percutaneous epididymal spermaspiration for obstructive azoospermia. Hum. Reprod., 11, 2058-2061.

Silber, S.J., Ord, T., Balmaceda, J. et al. (1990) Congenital absence of the vas deferens: thefertilizing capacity of human epididymal sperm. N. Eng. J. Med., 323, 1788-1792.

Silber, S., Nagy, Z., Liu, J. et al. (1994) Conventional in-vitro fertilization versus intracytoplasmicsperm injection for patients requiring microsurgical sperm aspiration. Hum. Reprod., 9, 1705-1709.

Silber, S.J., Nagy, Z., Liu, J. et al. (1995a) The use of epididymal and testicular spermatozoa forintracytoplasmic sperm injection: The genetic implications for male infertility. Hum. Reprod.,10, 2031-2043.

Silber, S.J., Van Steirteghem, A.C. and Devroey, P. (1995b) Sertoli cell only revisited. Hum.Reprod., 10, 1031-1032.

Silber, S.J., Van Steirteghem, A.C, Liu, J. et al. (1995c) High fertilization and pregnancy rateafter intracytoplasmic sperm injection with spermatozoa obtained from testicle biopsy. Hum.Reprod., 10, 148-152.

Silber, S.J., Van Steirteghem, A., Nagy, Z. et al. (1996) Normal pregnancies resulting fromtesticular sperm extraction and intracytoplasmic sperm injection for azoospermia due tomaturation arrest. Fertil. Steril., 66, 110-117.

Silber, S., Nagy, Z., Devroey, P. et al. (1997a) The effect of female age and ovarian reserve onpregnancy rate in male infertility: treatment of azoospermia with sperm retrieval andintracytoplasmic sperm injection. Hum. Reprod., 12, 2693-2700.

Silber, S., Nagy, Z., Devroey, P. et al. (1997b) Distribution of spermatogenesis in the testicles ofazoospermic men: the presence or absence of spermatids in the testes of men with germinalfailure. Hum. Reprod., 12, 2422-2428.

Staessen, C, Coonen, E., Van Assche, E. et al. (1996) Preimplantation diagnosis for X and Ynormality in embryos from three Klinefelter patients. Hum. Reprod., 11, 1650-1653.

Staessen, C, Van Assche, E., Joris, H. et al. (1997) Preimplantation diagnosis by fluorescent in-situ 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-vitrofertilisation (IVF) using sperm aspirated from the epididymis. / . In Vitro Fertil. Embryo Transfer,2, 112-122.

Tournaye, H., Devroey, P., Liu, J. et al. (1994) Microsurgical epididymal sperm aspiration andintracytoplasmic sperm injection: a new effective approach to infertility as a result of congenitalbilateral absence of the vas deferens. Fertil. Steril., 61, 1045-1051.

Tournaye, H., Camus, M., Goossens, A. et al. (1995) Recent concepts in the management ofinfertility because of non-obstructive azoospermia. Hum. Reprod., 10 (Suppl. 1), 115-119.

Tournaye, H., Liu, J., Camus, M. et al. (1996a) Correlation between testicular histology andoutcome after intracytoplasmic sperm injection using testicular spermatozoa. Hum. Reprod., 11,127-132.

Tournaye, H., Staessen, C, Liebaers, I. et al. (1996b) Testicular sperm recovery in nine 47,XXYKlinefelter patients. Hum. Reprod., 11, 1644-1649.

Tournaye, H., Verheyen, G., Nagy, P. et al. (1997) Are there any predictive factors for successfultesticular sperm recovery in azoospermic patients? Hum. Reprod., 12, 80-86.

Tsirigotis, M. and Craft, I. (1995) Sperm retrieval methods and ICSI for obstructive azoospermia.Hum. Reprod., 10, 758-760.

Tsirigotis, M. and Craft, I. (1996) Micro-epididymal sperm aspiration or percutaneous epididymalsperm aspiration? The dilemma. Hum. Reprod., 11, 680-681.

Tsirigotis, M., Pelekanos, M., Beski, S. et al. (1996) Cumulative experience of percutaneous59



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A.Safran et al.

epididymal sperm aspiration (PESA) with intracytoplasmic sperm injection. J. Assist. Reprod.Genet., 13, 315-319.

Van Steirteghem, A.C., Nagy, Z., Joris, H. et al. (1993) High fertilization and implantation ratesafter intracytoplasmic sperm injection. Hum. Reprod., 8, 1061-1966.

Watkins, W., Nieto, E, Bourne, H. et al. (1997) Testicular and epididymal sperm in a microinjectionprogram: methods of retrieval and results. Fertil. Steril., 67, 527-535.

Yemini, M., Vanderzwalmen, P., Mukaida, T. et al. (1995) Intracytoplasmic sperm injection,fertilization and embryo transfer after retrieval of spermatozoa by testicular biopsy from anazoospermic male with testicular tubular atrophy. Fertil. Steril., 63, 1118-1120.

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