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Testicular sperm retrieval for assisted reproductive techniques
Reviews in Gynaecological Practice 4 (2004) 105–110
Testicular sperm retrieval for assisted reproductive techniques S.K.H. Ashour∗ , P.S.R. Murthy Assisted Reproduction Unit, Al-Mana General Hospital, Dammam 31451, P.O. Box 2366, Saudi Arabia Received 15 December 2003; accepted 1 December 2004 Available online 14 March 2004
Abstract The possibility of achieving a pregnancy with just one spermatozoon has led to the evolution of a number of methods aiming to obtain sperm from testicular tissue. This achievement now offers men with azoospermia a reasonable chance of fathering their own children. The choice of the best testicular sperm retrieval technique has always been a challenge. There has been a trend to use percutaneous needle aspiration but controlled studies have demonstrated that open testicular biopsy is necessary to obtain the best results, particularly in non-obstructive azoospermia. Since azoospermic men are at risk of specific genetic defects, careful genetic testing and counselling prior to treatment should be considered to allow the couple to make an informed decision as to whether or not to use the husband sperm. The recovery of sperm may not always be successful particularly in non-obstructive azoospermic men, an event that encompasses important emotional and financial implications. Unfortunately, no clinical or laboratory test can clearly predict success with sperm retrieval procedures. © 2004 Elsevier B.V. All rights reserved. Keywords: Male infertility; Azoospermia; Intracytoplasmic sperm injection; Testicular sperm retrieval
Twenty-five years ago, a fruitful collaboration between Steptoe and Edwards resulted in the birth of the first human baby after in vitro fertilization (IVF) and subsequent embryo transfer [1] Their report represented a major breakthrough in human infertility treatment, and IVF has since then become a well-established procedure for certain types of infertility. It soon, however, became obvious that couples with male factor infertility could not be helped by conventional IVF. The most severe expression of male factor infertility is azoospermia, in which the ejaculate is devoid of sperm. Causes of azoospermia include congenital and acquired reproductive tract obstruction and testicular failure. Intracytoplasmic sperm injection (ICSI), introduced by Palermo et al. [2] in 1992, has allowed this problem to be overcome. This approach involves the in vitro injection of a single sperm into each oocyte during an IVF cycle. ICSI essentially bypasses all natural barriers to fertilization, such as sperm interaction with the zona pellucida and sperm–egg fusion [2]. The ability to retrieve sperm from the male genital tract, combined with ICSI, has revolutionized the treatment of male factor infertility. For men with irreparable obstructive azoospermia, sperm retrieval is nearly certain and provides an excellent chance of conception. Refinements in sperm retrieval techniques now allow the treatment ∗ Corresponding
author. Tel.: +966-0538-37170. E-mail address: [email protected] (S.K.H. Ashour).
1471-7697/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.rigp.2004.01.004
of non-obstructive azoospermia, i.e. patients with primary testicular failure [3–5]. This chapter reviews the literature on testicular sperm retrieval, different retrieval techniques, evaluation of the results, complications and prognostic indicators of success.
1. Assessment Before considering sperm retrieval for a couple with a male partner with azoospermia, it is important to determine whether lack of sperm in the ejaculate is caused by an obstructive or a non-obstructive process. To an experienced examiner, the history and physical examination can suggest the cause of azoospermia. For example, the presence of normal-volume testes with bilaterally indurated epididymes or absent vasa deferentia points to an obstructive cause. A history of cryptorchidism in the presence of small-volume or soft testes may suggest non-obstructive azoospermia, especially if supported by an elevated follicle-stimulating hormone (FSH) level. In most cases, bilateral testicular biopsy or testicular aspiration will be indicated to confirm the impression of obstructive or non-obstructive azoospermia. Careful evaluation of the post-ejaculate urinalysis is necessary to rule out retrograde ejaculation as these men do not require invasive procedures for sperm retrieval; sperm may be isolated from the urine
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or via a catheter from the bladder and used for intrauterine insemination or IVF. In some cases of obstructive azoospermia, sperm retrieval can be achieved through the epididymis by either the percutaneous (PESA) or microsurgical (MESA) route. In other instances, sperm retrieval can be carried out through the vas deferens. In such cases, the sperm yield and sperm quality are superior to those of testicular sperm. Testicular sperm retrieval is indicated for all cases of non-obstructive azoospermia and some cases of obstructive azoospermia in which the expertise or facilities for PESA or MESA are not available. Men with obstructive azoospermia have a higher success rate for sperm retrieval and are at risk of carrying genetic defects different from those seen in men with non-obstructive azoospermia. Bilateral vasal agenesis is the most common congenital anomaly of the male reproductive tract that causes obstructive azoospermia. Congenital bilateral absence of the vas deferens is found in 1–2% of the infertile male population; in 42–66% of men with this defect, one or more cystic fibrosis gene mutations will be detectable. Conversely, almost all men with cystic fibrosis manifest infertility because of bilaterally absent vasa. In addition, up to 47% of men with idiopathic epididymal obstruction are known to be carriers of cystic fibrosis gene mutations [6]. The female partner should also be tested for cystic fibrosis gene mutations prior to attempts at conception with sperm retrieval. Different genetic defects should be considered in men with non-obstructive azoospermia. Up to 13% or more of men with this condition will have microdeletions of the Y chromosome, including deletions of the DAZ (deleted in azoospermia) gene [7]. Because of the likelihood of transmitting these mutations of the Y chromosome to the male offspring, it is important to address the whole issue of pre-operative screening and genetic counselling for men who are about to undergo testicular sperm retrieval. Testicular sperm retrieval combined with ICSI has in fact led to sterile men having male children who are also probably likely to be sterile. Severe male infertility or sterility resulting from genetic mutations would previously have ended with the sterile father, but we can be relatively certain that a father’s infertility will now be transmitted to his male offspring. Rucker et al. have shown testing the Y chromosome is not the only aspect of genetic counselling that these patients must consider. Many such azoospermic men will be discovered to have a sex chromosomal abnormality, such as Klinefelter syndrome (XXY). More importantly, a small percentage (approximately 1.5%) of azoospermic or severely oligospermic men will be discovered on chromosomal analysis to have balanced translocations, an incidence approximately 7–10 times that found in a fertile male population [8,9] This finding should not, however, cause alarm: Rucker et al. and others have clearly shown that the Klinefelter XXY anomaly does not appear to lead to children with Klinefelter syndrome [10–12]. An awareness of the karyotype will be an important part of counselling these patients on the advis-
ability of pre-implantation genetic diagnosis, amniocentesis or chorionic villus sampling if they are concerned about the low risk of chromosomal error in the offspring. The evidence is actually more reassuring than alarming in that the incidence of congenital abnormality in ICSI children has been no greater than in every normal population studied (2.3%), and the chromosomal abnormalities in the offspring have appeared not to occur with any greater frequency than in the normal population, except for a slightly higher incidence of Klinefelter syndrome and other sex chromosomal abnormalities (0.8%) [8,9]. There should therefore be no cause for alarm because, with the exception of the transmission of infertility to the offspring, newborns are for the most part as likely to be normal as those in a fertile population.
2. Predictors of success Although testicular sperm retrieval is not difficult in cases of obstructive azoospermia, it is successful in only 50% of cases of non-obstructive azoospermia [13]. An unsuccessful sperm recovery procedure has important emotional and financial implications, as well as complications such as testicular devascularization and fibrosis [14,15] Unfortunately, no clinical or laboratory feature can clearly predict the success of sperm retrieval procedures [3,4,16,18]. Serum FSH level and testicular volume can be used to determine whether sperm production is normal (as in obstructive azoospermia) or severely abnormal (as in non-obstructive azoospermia). The use of these parameters to predict the success of sperm retrieval in men with non-obstructive azoospermia has, however, been disappointing [4,5]. In men with complete maturational arrest, for example, normal testicular volumes and normal FSH levels may be seen but no retrievable sperm may be evident. In contrast, a man with predominately Sertoli cell-only testes but few focal areas of normal spermatogenesis is likely to have a smaller testicular volume and an elevated FSH level, with many spermatozoa retrievable on testicular sperm extraction. Although none of the above-mentioned parameters has proved highly predictive, diagnostic testicular biopsy is repeatedly reported to be a valuable predictor of successful testicular sperm retrieval in non-obstructive azoospermia [16,18–21]. However, even the absence of sperm in a single testicular biopsy does not guarantee a complete lack of sperm in the testes [13,22]. Serum inhibin B, a direct product of the Sertoli cells, is another marker of spermatogenesis, but its role as a predictor of finding sperm on testicular sperm retrieval is still controversial [23–26]. In two studies, inhibin B has had a clear predictive value for finding sperm on surgical testicular sperm retrieval [27] or diagnostic testicular biopsy [28]. Others have concluded that inhibin B alone or in combination with FSH is of limited predictive value in successful sperm retrieval [29].
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3. Techniques The goals of testicular sperm retrieval are to confirm the presence of sperm prior to IVF/ICSI, to retrieve an adequate number of sperm for both immediate use and (where possible) cryopreservation, and to minimize damage to the reproductive tract in order not to jeopardize future attempts at sperm retrieval or testicular function. Several techniques have been described to try to achieve these goals.
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23-gauge, fine butterfly needle connected to a 5 ml syringe placed on suction to create a negative pressure. Precise, gentle in-and-out movements, varying in length from 5 to 8 mm, are used to aspirate the tissue fragments. The suction is then released, and the tissue fragments are expelled on to a small dish. These are immediately sent for examination by an experienced cytologist. Pressure is then applied to the aspiration site to obtain haemostasis. 3.2. Open testicular sperm extraction
3.1. Percutaneous testicular sperm aspiration (TESA) Testicular fine-needle aspiration is an established technique for the diagnosis and treatment of male infertility [30,31]. This technique was initially described as a diagnostic procedure in azoospermic men. Subsequently, testicular fine-needle aspiration for the recovery of spermatozoa prior to IVF/ICSI has been described [32]. The advantages of percutaneous aspiration techniques are that they can be performed with less anaesthesia, without open scrotal exploration and its attendant post-operative discomfort, and without microsurgical or urosurgical expertise. Percutaneous techniques are easily repeatable as well as potentially less expensive than microsurgical techniques and do not require special microsurgical training; they are also easily performed by a gynaecologist in the absence of a trained urologist. The inconsistent ability to retrieve an adequate number and quality of sperm for cryopreservation with percutaneous approaches is a disadvantage compared with open sperm retrieval. In addition, occasional haematoceles and haematomas may occur after percutaneous sperm retrieval. Attempts at IVF and ICSI with testicular sperm derived from atrophic, non-obstructed testes have revealed not only that small, non-obstructive azoospermic testes can harbour sperm but also that spermatogenesis varies geographically in such testes, which might lead to a failure of sperm retrieval and a cancellation of IVF cycles, at great expense to the couple [3,4,14,17]. To tackle this, a refined modification of testicular aspiration, called fine-needle aspiration mapping, has been introduced into the evaluation of infertile, azoospermic men ([33,34]). This provides information on the state of spermatogenesis in all regions of the testis and assists in subsequent sperm retrieval by directing biopsy to sperm-rich areas. As a testis-sparing procedure, it minimizes the removal of valuable hormone-producing tissue that is required for normal endocrine functions. 3.1.1. Aspiration technique After local or general anaesthesia has been administered, the testis is positioned with the epididymis directed posteriorly and the scrotal skin stretched over the testis by wrapping it behind the testis with a sponge. The testicular wrap will serve as a convenient handle to manipulate the testis and also to fix the scrotal skin over the testis during the procedure. Fine-needle aspiration is performed with a sharp-bevelled,
Testicular biopsy is mainly performed in infertile men to distinguish between obstructive and non-obstructive azoospermia. Surgical sperm retrieval via open testis biopsy can yield viable sperm in nearly 100% of cases of obstructive azoospermia, although it yields viable sperm in only 60% of men with non-obstructive azoospermia [35]. In addition, the histology on diagnostic biopsy can predict successful testicular sperm retrieval from men with nonobstructive azoospermia [19]. In these men, the goal is to identify and retrieve sperm-bearing testicular tissue, which often requires multiple biopsies and the removal of larger samples of testis than those retrieved for diagnostic biopsy alone. Excising seminiferous tubules devoid of germinal elements that are otherwise hormonally active has several disadvantages. Primary hypogonadism may potentially be produced if a considerable number of Leydig cells are removed [36]. Multiple biopsies also endanger the testicular blood supply and risk devascularization of the testicle [15]. In addition, human autopsy studies of testicular blood supply have shown that no single area of the tunica albuginea can be blindly opened without potential injury to a major vessel of the testis, again risking testicular devascularization [37]. Harrington et al. [38] have reported that 29% of single open diagnostic testicular biopsies caused intratesticular haematoma formation, as detected on ultrasound. 3.3. Microsurgical testicular biopsy This procedure is performed with the patient under general or regional anaesthesia. An operating microscope providing a magnification of between 6× and 25× is used for testicular biopsy. Under 6× magnification, a 1 cm transverse skin incision is made overlying the mid-portion of the testis, with the epididymis fixed posteriorly. The tunica vaginalis is exposed, entered and held open with clamps. A median raphe or large transverse incision is made, and each testicle is delivered as needed. The tunica albuginea is then inspected under 15× magnification. Tunica perforating arteries from the testicular artery and vasal artery enter medially at the poles and course immediately below the tunica albuginea before branching and disappearing in the parenchyma as end-arteries. Subtunical vessels can easily be seen and avoided. Areas of high vessel concentration within the parenchyma can be seen with
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the microscope through the tunica as blue or red areas and avoided when making the incision in the tunica. An avascular region near the mid-portion of the anterior surface is chosen for the tunical incision. Care is taken while making the incision not to extend it deeply into the parenchyma where the major arterial vessels are located. A 15◦ ultrasharp ophthalmic knife is used to make an incision in the tunica that is approximately 5–10 mm long. Subtunical crossing vessels are cauterized before excising tissue with a bipolar cautery. An attempt is made to locate large white or yellow tubules, which are more likely to contain sperm than are the small brown or tan tubules [39]. If needed, the incision is extended and the testicular parenchyma is inspected closely to find tubules likely to contain sperm, which are excised with sharp iris scissors. Small bleeders are controlled with a bipolar cautery. The seminiferous tubules are examined under the light microscope without staining and are graded for concentration of spermatozoa, motility, percentage progression and percentage morphology. If no sperm are found, biopsy is performed on the opposite testis. If still no sperm are found, repeat biopsies are taken from different locations on both testes. The tunica albuginea and tunica vaginalis incisions are closed with 5/0 polyglactin sutures and the skin with a subcuticular 5/0 poliglecaprone suture. This microsurgical approach to testicular sperm extraction is particularly useful in cryptorchid azoospermia or Klinefelter syndrome, when fibrosis is interspersed with tubules possessing normal spermatogenesis. In these cases, it is relatively easy with the microsurgical approach to remove the tubules that have spermatogenesis and thereby conserve testicular tissue. It is much more difficult to accomplish this selection in cases of maturation arrest when all tubules show spermatogenesis and spermatogenesis goes to completion only in occasional tubules. Even in those cases, however, the microsurgical approach to testicular sperm extraction will, if necessary, allow the removal of larger amounts of testicular tissue with a minimum amount of damage and secondary testicular atrophy. With these microsurgical improvements in testicular sperm extraction, a much greater success rate will be achieved for non-obstructive azoospermia than is possible with blind needle aspiration. The microsurgical technique is safe and effective when sampling testicular tissue for diagnostic or therapeutic purposes. Compared with non-microscopic open biopsies, this technique reduces the incidence of complications and maximizes the ability to take multiple samples safely. It also appears to help in the selection of tubules most likely to contain sperm in men with non-obstructive azoospermia [39].
4. Results Most series have indicated that there are no individual characteristics of the men themselves that will further predict the chance of achieving pregnancy after a sperm retrieval
procedure. There does appear to be a difference between results for obstructive and non-obstructive azoospermia, partly because not all men with non-obstructive azoospermia have retrievable sperm. 4.1. Obstructive azoospermia Pregnancy rates after sperm retrieval will depend on the certainty with which the diagnosis of obstruction has been made prior to retrieval as well as on the overall results achieved at that IVF unit. Belker et al. [40] reported a clinical pregnancy rate of 40% when testicular sperm retrieval was performed using percutaneous fine-needle aspiration, whereas Gil-Salom et al. [41] described a 27% pregnancy rate with open testicular biopsy. 4.2. Non-obstructive azoospermia The results of sperm retrieval by open testicular biopsy for men with non-obstructive azoospermia are made up of two different components. The first is the ability to retrieve sperm from the testis, the second is the pregnancy rate after ICSI using those sperm. One of the factors that may reduce the chance of sperm retrieval is repeating the testicular biopsy within 6 months; this also increases the risk of permanent testicular devascularization [5]. Reports of the chance of retrieving sperm have been as high as 90% for men without obstruction in a small series and as low as 50% in larger series. Obviously, the more carefully the men are selected before retrieval, the better is the likelihood of finding sperm with TESE. One team initially reported that 80% of men had sperm retrieved with TESE. The fertilization rate per injected oocyte was 48%, with an overall clinical pregnancy rate of 20% per attempt at TESE [3]. Subsequent results from Belgium suggest that the rate of sperm retrieval is closer to 50% per TESE procedure [4]. Another group reported that 10 out of 16 (62%) men without obstruction had sperm extracted with TESE. The fertilization rate per injected oocyte was 58%, and 5 out of 10 (50%) of those undergoing ICSI achieved a clinical pregnancy rate of 31% per attempt at TESE [42].
5. Summary Testicular sperm retrieval from men with azoospermia is now possible with acceptable-to-excellent pregnancy rates when used alongside ICSI. Advances in both sperm retrieval and assisted reproduction have widened the potential of fertility treatment when the only management options a few years previously had been donor insemination and adoption. Extensive multiple biopsies from every area of the testis are often performed in an effort to find sufficient spermatozoa for ICSI. This can result in a great deal of testicular damage and an alteration in testicular function, including testicular atrophy. This damage can be minimized by using
S.K.H. Ashour, P.S.R. Murthy / Reviews in Gynaecological Practice 4 (2004) 105–110
needle rather than open biopsy to obtain spermatozoa for ICSI. This is possible and has proved to be successful in cases of obstructive azoospermia; however, when spermatogenesis is deficient, needle biopsy is much less likely than open biopsy to find the rare foci of spermatogenesis necessary for ICSI. Since our ultimate goal is to reduce the amount of testicular tissue removed, it has been noted that the scarce foci of normal spermatogenesis in cases of non-obstructive azoospermia could be detected directly under the operating microscope. Only tiny portions of testicle would then need to be removed using this microsurgical technique. Another advance is the ability to use cryopreserved testicular spermatozoa, which will limit the number of sperm retrieval procedures necessary to achieve fertility for a couple. Specific genetic abnormalities are associated with both types of azoospermia, and a careful evaluation of the cause of azoospermia and genetic counselling are indicated for all azoospermic men. A significant proportion of non-obstructive azoospermic men will fail to have sperm retrieved with testicular sperm extraction procedures. To date, there has been no good prognostic indicator of sperm retrieval success in such cases. The most sensitive markers are currently fine-needle aspiration mapping and open testicular biopsy. The search should continue to find more reliable indicators. The choice of a suitable testicular sperm retrieval technique will depend on the aetiology of the azoospermia, the therapeutic purpose of the procedure, the expertise of the operator and the available facilities. Acknowledgements The authors thank Dr. Dee Kini FRCOG, the director of the IVF unit at the Al-Mana General Hospital, Dammam, for his assistance in the preparation of this manuscript. References [1] Steptoe PC, Edwards RG. Birth after the re-implantation of a human embryo. Lancet 1978;2:366. [2] Palermo GD, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992;340:17. [3] Devroey P, Liu J, Nagy Z, et al. Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermia. Hum Reprod 1995;10:1457. [4] Kahraman S, Ozgur S, Alatas C, et al. Fertility with testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermic men. Hum Reprod 1996;11:756. [5] Schlegel PN, Palermo GD, Goldstein M, et al. Testicular sperm extraction with intracytoplasmic sperm injection for nonobstructive azoospermia. Urology 1997;49:435. [6] Girardi SK, Schlegel PN. Microsurgical epididymal sperm aspiration: review of techniques, preoperative considerations, and results. J Androl 1996;1:5. [7] Reijo R, Lee T, Salo P, et al. Diverse spermatogenic defect in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nat Genet 1995;10:383.
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[8] Bonduelle M, Wilikens A, Buysse A, et al. Prospective follow-up study of 877 children born after intracytoplasmic sperm injection (ICSI), with ejaculated epididymal and testicular spermatozoa and after replacement of cryopreserved embryos obtained after ICSI. Hum Reprod 1996;4(Suppl):131. [9] Bonduelle M, Aytoz A, Wilikens A, et al. Prospective follow-up study of 1,987 children born after intracytoplasmic sperm injection (ICSI). In: Filicori M, Flamigni C, editors. Treatment of infertility: the new frontiers. New Jersey: Communications Media for Education; 1998. p. 445–61. [10] Tournaye H, Staessen C, Liebaers I. Testicular sperm recovery in nine 47,XXY Klinefelter patients. Hum Reprod 1996;11:1644. [11] Staessen C, Coonen E, Van Assche E, et al. Preimplantation diagnosis for X and Y normality in embryos from three Klinefelter patients. Hum Reprod 1996;11:1650. [12] Palermo GD, Schlegel PN, Sills ES, et al. Births after intracytoplasmic injection of sperm obtained by testicular extraction from men with nonmosaic Klinefelter’s syndrome. N Engl J Med 1998;338:588. [13] Tournaye H, Camus M, Goossens A, et al. Recent concepts in the management of infertility because of non-obstructive azoospermia. Hum Reprod 1995;10(Suppl 1):115–9. [14] Friedler S, Raziel A, Strassburger D, Soffer Y, Komarovsky D, Ron-El R. 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 1997;12:1488–93. [15] Schlegel PN, Su L. Physiological consequences of testicular sperm extraction. Hum Reprod 1997;12:1688–92. [16] Tournaye H, Liu J, Nagy PZ, et al. Correlation between testicular histology and outcome after intracytoplasmic sperm injection using testicular spermatozoa. Hum Reprod 1996;11:127. [17] Kim ED, Gilbaugh Jr JH, Patel VR, et al. Testis biopsies frequently demonstrate sperm in men with azoospermia and significantly elevated follicle-stimulating hormone levels. J Urol 1997;157:144. [18] Gil-Salom M, Romero J, Minguez Y, Molero MD, Remohi J, Pellicer A. Testicular sperm extraction and intracytoplasmic sperm injection: chance of fertility in nonobstructive azoospermia. J Urol 1998;160:2063. [19] Su LM, Palermo GD, Goldstein M, Veeck LL, Rosenwaks Z, Schlegel PN. Testicular sperm extraction with intracytoplasmic sperm injection for nonobstructive azoospermia: testicular histology can predict success of sperm retrieval. J Urol 1999;161:112. [20] Silber SJ, Nagy Z, Devroey P, Tournaye H, Van Steirteghem AC. 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 1997;12:2422. [21] Plas E, Pflüger H. Value of testicular biopsy prior to TESE and ICSI in patients with non-obstructive azoospermia. Int J Androl 1997;20(Suppl):36. [22] Gottschalk-Sabag S, Weiss DB, Folb-Zacharow N, Zukerman Z. Is one testicular specimen sufficient for quantitative evaluation of spermatogenesis? Fertil Steril 1995;64:399–402. [23] Anawalt BD, Bebb RA, Matsumoto, et al. Serum inhibin B levels reflect Sertoli cell function in normal men and men with testicular dysfunction. J Clin Endocrinol Metab 1996;81:3341–5. [24] Illingworth PJ, Groome NP, Byrd W, et al. Inhibin-B: a likely candidate for the physiologically important form of inhibin in men. J Clin Endocrinol Metab 1996;81:1321–5. [25] Jensen TK, Andersson A-M, Hjollund NHI, et al. Inhibin B as a serum marker of spermatogenesis: correlation to differences in sperm concentration and follicle-stimulating hormone levels. A study of 349 Danish men. J Clin Endocrinol Metab 1997;82:4059–63. [26] Kolb BA, Stanczyk FZ, Sokol RZ. Serum inhibin B levels in males with gonadal dysfunction. Fertil Steril 2000;74:234–8. [27] Ballesca JL, Balasch J, Calafell JM, et al. Serum inhibin B determination is predictive of successful testicular sperm extraction in men with non-obstructive azoospermia. Hum Reprod 2000;15:1734–8.
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[28] Pierik FH, Vreeburg JTM, Stijnen T, De Jong FH, Weber RFA. Serum inhibin B as a marker of spermatogenesis. J Clin Endocrinol Metab 1998;83:3110–4. [29] Von Eckardstein S, Simoni M, Bergmann M, et al. Serum inhibin B in combination with serum follicle-stimulating hormone (FSH) is a more sensitive marker than serum FSH alone for impaired spermatogenesis in men, but cannot predict the presence of sperm in testicular tissue samples. J Clin Endocrinol Metab 1999;84:2496– 501. [30] Mallidis C, Baker HW. Fine needle tissue aspiration biopsy of the testis. Fertil Steril 1994;61:367. [31] Mahajan AD, Ali NI, Walwalkar SJ, et al. The role of fine-needle aspiration cytology of the testis in the diagnostic evaluation of infertility. BJU Int 1999;84:485. [32] Bourne H, Watkins W, Speirs A, et al. Pregnancies after intracytoplasmic injection of sperm collected by fine needle biopsy of the testis. Fertil Steril 1995;64:433. [33] Turek PJ, Cha I, Ljung B-M. Systematic fine-needle aspiration of the testis: correlation to biopsy and results of organ ‘mapping’ for mature sperm in azoospermic men. Urology 1997;49:743. [34] Turek PJ, Givens CR, Schriock ED, et al. Testis sperm extraction and intracytoplasmic sperm injection guided by prior fine-needle aspiration mapping in patients with nonobstructive azoospermia. Fertil Steril 1999;71:552.
[35] Ben-Yosef D, Yogev L, Hauser R, et al. Testicular sperm retrieval and cryopreservation prior to initiating ovarian stimulation as the first line approach in patients with non-obstructive azoospermia. Hum Reprod 1999;14:1794. [36] Manning M, Junemann KP, Alken P. Decrease in testostrone blood concentrations after testicular sperm extraction for intracytoplasmic sperm injection in azoospermic men. Lancet 1998;352:37. [37] Jarow JP. Clinical significance of intratesticular arterial anatomy. J Urol 1991;145:777. [38] Harrington TG, Schauer D, Gilbert BR. Percutaneous testis biopsy: an alternative to open testicular biopsy in evaluation of the subfertile man. J Urol 1996;156:1647. [39] Schlegel PN. Testicular sperm extraction: microdissection improves sperm yield with minimal tissue excision. Hum Reprod 1999;14: 131. [40] Belker AM, Sherins RJ, Coulan CB, et al. High fertilization and pregnancy rates obtained by nonsurgical percutaneous needle aspiration of testicular sperm. J Urol 1996;155:364. [41] Gil-Salom M, Minguez Y, Rubio C, et al. Intracytoplasmic testicular sperm injection: an effective treatment for otherwise intractable obstructive azoospermia. J Urol 1995;154:2974. [42] Schlegel PN, Palermo GD, Goldstein M, et al. Testicular sperm extraction with ICSI for nonobstructive azoospermia. Urology 1997;49(3):435–40.
Testicular sperm retrieval for assisted reproductive techniques S.K.H. Ashour∗ , P.S.R. Murthy Assisted Reproduction Unit, Al-Mana General Hospital, Dammam 31451, P.O. Box 2366, Saudi Arabia Received 15 December 2003; accepted 1 December 2004 Available online 14 March 2004
Abstract The possibility of achieving a pregnancy with just one spermatozoon has led to the evolution of a number of methods aiming to obtain sperm from testicular tissue. This achievement now offers men with azoospermia a reasonable chance of fathering their own children. The choice of the best testicular sperm retrieval technique has always been a challenge. There has been a trend to use percutaneous needle aspiration but controlled studies have demonstrated that open testicular biopsy is necessary to obtain the best results, particularly in non-obstructive azoospermia. Since azoospermic men are at risk of specific genetic defects, careful genetic testing and counselling prior to treatment should be considered to allow the couple to make an informed decision as to whether or not to use the husband sperm. The recovery of sperm may not always be successful particularly in non-obstructive azoospermic men, an event that encompasses important emotional and financial implications. Unfortunately, no clinical or laboratory test can clearly predict success with sperm retrieval procedures. © 2004 Elsevier B.V. All rights reserved. Keywords: Male infertility; Azoospermia; Intracytoplasmic sperm injection; Testicular sperm retrieval
Twenty-five years ago, a fruitful collaboration between Steptoe and Edwards resulted in the birth of the first human baby after in vitro fertilization (IVF) and subsequent embryo transfer [1] Their report represented a major breakthrough in human infertility treatment, and IVF has since then become a well-established procedure for certain types of infertility. It soon, however, became obvious that couples with male factor infertility could not be helped by conventional IVF. The most severe expression of male factor infertility is azoospermia, in which the ejaculate is devoid of sperm. Causes of azoospermia include congenital and acquired reproductive tract obstruction and testicular failure. Intracytoplasmic sperm injection (ICSI), introduced by Palermo et al. [2] in 1992, has allowed this problem to be overcome. This approach involves the in vitro injection of a single sperm into each oocyte during an IVF cycle. ICSI essentially bypasses all natural barriers to fertilization, such as sperm interaction with the zona pellucida and sperm–egg fusion [2]. The ability to retrieve sperm from the male genital tract, combined with ICSI, has revolutionized the treatment of male factor infertility. For men with irreparable obstructive azoospermia, sperm retrieval is nearly certain and provides an excellent chance of conception. Refinements in sperm retrieval techniques now allow the treatment ∗ Corresponding
author. Tel.: +966-0538-37170. E-mail address: [email protected] (S.K.H. Ashour).
1471-7697/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.rigp.2004.01.004
of non-obstructive azoospermia, i.e. patients with primary testicular failure [3–5]. This chapter reviews the literature on testicular sperm retrieval, different retrieval techniques, evaluation of the results, complications and prognostic indicators of success.
1. Assessment Before considering sperm retrieval for a couple with a male partner with azoospermia, it is important to determine whether lack of sperm in the ejaculate is caused by an obstructive or a non-obstructive process. To an experienced examiner, the history and physical examination can suggest the cause of azoospermia. For example, the presence of normal-volume testes with bilaterally indurated epididymes or absent vasa deferentia points to an obstructive cause. A history of cryptorchidism in the presence of small-volume or soft testes may suggest non-obstructive azoospermia, especially if supported by an elevated follicle-stimulating hormone (FSH) level. In most cases, bilateral testicular biopsy or testicular aspiration will be indicated to confirm the impression of obstructive or non-obstructive azoospermia. Careful evaluation of the post-ejaculate urinalysis is necessary to rule out retrograde ejaculation as these men do not require invasive procedures for sperm retrieval; sperm may be isolated from the urine
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or via a catheter from the bladder and used for intrauterine insemination or IVF. In some cases of obstructive azoospermia, sperm retrieval can be achieved through the epididymis by either the percutaneous (PESA) or microsurgical (MESA) route. In other instances, sperm retrieval can be carried out through the vas deferens. In such cases, the sperm yield and sperm quality are superior to those of testicular sperm. Testicular sperm retrieval is indicated for all cases of non-obstructive azoospermia and some cases of obstructive azoospermia in which the expertise or facilities for PESA or MESA are not available. Men with obstructive azoospermia have a higher success rate for sperm retrieval and are at risk of carrying genetic defects different from those seen in men with non-obstructive azoospermia. Bilateral vasal agenesis is the most common congenital anomaly of the male reproductive tract that causes obstructive azoospermia. Congenital bilateral absence of the vas deferens is found in 1–2% of the infertile male population; in 42–66% of men with this defect, one or more cystic fibrosis gene mutations will be detectable. Conversely, almost all men with cystic fibrosis manifest infertility because of bilaterally absent vasa. In addition, up to 47% of men with idiopathic epididymal obstruction are known to be carriers of cystic fibrosis gene mutations [6]. The female partner should also be tested for cystic fibrosis gene mutations prior to attempts at conception with sperm retrieval. Different genetic defects should be considered in men with non-obstructive azoospermia. Up to 13% or more of men with this condition will have microdeletions of the Y chromosome, including deletions of the DAZ (deleted in azoospermia) gene [7]. Because of the likelihood of transmitting these mutations of the Y chromosome to the male offspring, it is important to address the whole issue of pre-operative screening and genetic counselling for men who are about to undergo testicular sperm retrieval. Testicular sperm retrieval combined with ICSI has in fact led to sterile men having male children who are also probably likely to be sterile. Severe male infertility or sterility resulting from genetic mutations would previously have ended with the sterile father, but we can be relatively certain that a father’s infertility will now be transmitted to his male offspring. Rucker et al. have shown testing the Y chromosome is not the only aspect of genetic counselling that these patients must consider. Many such azoospermic men will be discovered to have a sex chromosomal abnormality, such as Klinefelter syndrome (XXY). More importantly, a small percentage (approximately 1.5%) of azoospermic or severely oligospermic men will be discovered on chromosomal analysis to have balanced translocations, an incidence approximately 7–10 times that found in a fertile male population [8,9] This finding should not, however, cause alarm: Rucker et al. and others have clearly shown that the Klinefelter XXY anomaly does not appear to lead to children with Klinefelter syndrome [10–12]. An awareness of the karyotype will be an important part of counselling these patients on the advis-
ability of pre-implantation genetic diagnosis, amniocentesis or chorionic villus sampling if they are concerned about the low risk of chromosomal error in the offspring. The evidence is actually more reassuring than alarming in that the incidence of congenital abnormality in ICSI children has been no greater than in every normal population studied (2.3%), and the chromosomal abnormalities in the offspring have appeared not to occur with any greater frequency than in the normal population, except for a slightly higher incidence of Klinefelter syndrome and other sex chromosomal abnormalities (0.8%) [8,9]. There should therefore be no cause for alarm because, with the exception of the transmission of infertility to the offspring, newborns are for the most part as likely to be normal as those in a fertile population.
2. Predictors of success Although testicular sperm retrieval is not difficult in cases of obstructive azoospermia, it is successful in only 50% of cases of non-obstructive azoospermia [13]. An unsuccessful sperm recovery procedure has important emotional and financial implications, as well as complications such as testicular devascularization and fibrosis [14,15] Unfortunately, no clinical or laboratory feature can clearly predict the success of sperm retrieval procedures [3,4,16,18]. Serum FSH level and testicular volume can be used to determine whether sperm production is normal (as in obstructive azoospermia) or severely abnormal (as in non-obstructive azoospermia). The use of these parameters to predict the success of sperm retrieval in men with non-obstructive azoospermia has, however, been disappointing [4,5]. In men with complete maturational arrest, for example, normal testicular volumes and normal FSH levels may be seen but no retrievable sperm may be evident. In contrast, a man with predominately Sertoli cell-only testes but few focal areas of normal spermatogenesis is likely to have a smaller testicular volume and an elevated FSH level, with many spermatozoa retrievable on testicular sperm extraction. Although none of the above-mentioned parameters has proved highly predictive, diagnostic testicular biopsy is repeatedly reported to be a valuable predictor of successful testicular sperm retrieval in non-obstructive azoospermia [16,18–21]. However, even the absence of sperm in a single testicular biopsy does not guarantee a complete lack of sperm in the testes [13,22]. Serum inhibin B, a direct product of the Sertoli cells, is another marker of spermatogenesis, but its role as a predictor of finding sperm on testicular sperm retrieval is still controversial [23–26]. In two studies, inhibin B has had a clear predictive value for finding sperm on surgical testicular sperm retrieval [27] or diagnostic testicular biopsy [28]. Others have concluded that inhibin B alone or in combination with FSH is of limited predictive value in successful sperm retrieval [29].
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3. Techniques The goals of testicular sperm retrieval are to confirm the presence of sperm prior to IVF/ICSI, to retrieve an adequate number of sperm for both immediate use and (where possible) cryopreservation, and to minimize damage to the reproductive tract in order not to jeopardize future attempts at sperm retrieval or testicular function. Several techniques have been described to try to achieve these goals.
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23-gauge, fine butterfly needle connected to a 5 ml syringe placed on suction to create a negative pressure. Precise, gentle in-and-out movements, varying in length from 5 to 8 mm, are used to aspirate the tissue fragments. The suction is then released, and the tissue fragments are expelled on to a small dish. These are immediately sent for examination by an experienced cytologist. Pressure is then applied to the aspiration site to obtain haemostasis. 3.2. Open testicular sperm extraction
3.1. Percutaneous testicular sperm aspiration (TESA) Testicular fine-needle aspiration is an established technique for the diagnosis and treatment of male infertility [30,31]. This technique was initially described as a diagnostic procedure in azoospermic men. Subsequently, testicular fine-needle aspiration for the recovery of spermatozoa prior to IVF/ICSI has been described [32]. The advantages of percutaneous aspiration techniques are that they can be performed with less anaesthesia, without open scrotal exploration and its attendant post-operative discomfort, and without microsurgical or urosurgical expertise. Percutaneous techniques are easily repeatable as well as potentially less expensive than microsurgical techniques and do not require special microsurgical training; they are also easily performed by a gynaecologist in the absence of a trained urologist. The inconsistent ability to retrieve an adequate number and quality of sperm for cryopreservation with percutaneous approaches is a disadvantage compared with open sperm retrieval. In addition, occasional haematoceles and haematomas may occur after percutaneous sperm retrieval. Attempts at IVF and ICSI with testicular sperm derived from atrophic, non-obstructed testes have revealed not only that small, non-obstructive azoospermic testes can harbour sperm but also that spermatogenesis varies geographically in such testes, which might lead to a failure of sperm retrieval and a cancellation of IVF cycles, at great expense to the couple [3,4,14,17]. To tackle this, a refined modification of testicular aspiration, called fine-needle aspiration mapping, has been introduced into the evaluation of infertile, azoospermic men ([33,34]). This provides information on the state of spermatogenesis in all regions of the testis and assists in subsequent sperm retrieval by directing biopsy to sperm-rich areas. As a testis-sparing procedure, it minimizes the removal of valuable hormone-producing tissue that is required for normal endocrine functions. 3.1.1. Aspiration technique After local or general anaesthesia has been administered, the testis is positioned with the epididymis directed posteriorly and the scrotal skin stretched over the testis by wrapping it behind the testis with a sponge. The testicular wrap will serve as a convenient handle to manipulate the testis and also to fix the scrotal skin over the testis during the procedure. Fine-needle aspiration is performed with a sharp-bevelled,
Testicular biopsy is mainly performed in infertile men to distinguish between obstructive and non-obstructive azoospermia. Surgical sperm retrieval via open testis biopsy can yield viable sperm in nearly 100% of cases of obstructive azoospermia, although it yields viable sperm in only 60% of men with non-obstructive azoospermia [35]. In addition, the histology on diagnostic biopsy can predict successful testicular sperm retrieval from men with nonobstructive azoospermia [19]. In these men, the goal is to identify and retrieve sperm-bearing testicular tissue, which often requires multiple biopsies and the removal of larger samples of testis than those retrieved for diagnostic biopsy alone. Excising seminiferous tubules devoid of germinal elements that are otherwise hormonally active has several disadvantages. Primary hypogonadism may potentially be produced if a considerable number of Leydig cells are removed [36]. Multiple biopsies also endanger the testicular blood supply and risk devascularization of the testicle [15]. In addition, human autopsy studies of testicular blood supply have shown that no single area of the tunica albuginea can be blindly opened without potential injury to a major vessel of the testis, again risking testicular devascularization [37]. Harrington et al. [38] have reported that 29% of single open diagnostic testicular biopsies caused intratesticular haematoma formation, as detected on ultrasound. 3.3. Microsurgical testicular biopsy This procedure is performed with the patient under general or regional anaesthesia. An operating microscope providing a magnification of between 6× and 25× is used for testicular biopsy. Under 6× magnification, a 1 cm transverse skin incision is made overlying the mid-portion of the testis, with the epididymis fixed posteriorly. The tunica vaginalis is exposed, entered and held open with clamps. A median raphe or large transverse incision is made, and each testicle is delivered as needed. The tunica albuginea is then inspected under 15× magnification. Tunica perforating arteries from the testicular artery and vasal artery enter medially at the poles and course immediately below the tunica albuginea before branching and disappearing in the parenchyma as end-arteries. Subtunical vessels can easily be seen and avoided. Areas of high vessel concentration within the parenchyma can be seen with
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the microscope through the tunica as blue or red areas and avoided when making the incision in the tunica. An avascular region near the mid-portion of the anterior surface is chosen for the tunical incision. Care is taken while making the incision not to extend it deeply into the parenchyma where the major arterial vessels are located. A 15◦ ultrasharp ophthalmic knife is used to make an incision in the tunica that is approximately 5–10 mm long. Subtunical crossing vessels are cauterized before excising tissue with a bipolar cautery. An attempt is made to locate large white or yellow tubules, which are more likely to contain sperm than are the small brown or tan tubules [39]. If needed, the incision is extended and the testicular parenchyma is inspected closely to find tubules likely to contain sperm, which are excised with sharp iris scissors. Small bleeders are controlled with a bipolar cautery. The seminiferous tubules are examined under the light microscope without staining and are graded for concentration of spermatozoa, motility, percentage progression and percentage morphology. If no sperm are found, biopsy is performed on the opposite testis. If still no sperm are found, repeat biopsies are taken from different locations on both testes. The tunica albuginea and tunica vaginalis incisions are closed with 5/0 polyglactin sutures and the skin with a subcuticular 5/0 poliglecaprone suture. This microsurgical approach to testicular sperm extraction is particularly useful in cryptorchid azoospermia or Klinefelter syndrome, when fibrosis is interspersed with tubules possessing normal spermatogenesis. In these cases, it is relatively easy with the microsurgical approach to remove the tubules that have spermatogenesis and thereby conserve testicular tissue. It is much more difficult to accomplish this selection in cases of maturation arrest when all tubules show spermatogenesis and spermatogenesis goes to completion only in occasional tubules. Even in those cases, however, the microsurgical approach to testicular sperm extraction will, if necessary, allow the removal of larger amounts of testicular tissue with a minimum amount of damage and secondary testicular atrophy. With these microsurgical improvements in testicular sperm extraction, a much greater success rate will be achieved for non-obstructive azoospermia than is possible with blind needle aspiration. The microsurgical technique is safe and effective when sampling testicular tissue for diagnostic or therapeutic purposes. Compared with non-microscopic open biopsies, this technique reduces the incidence of complications and maximizes the ability to take multiple samples safely. It also appears to help in the selection of tubules most likely to contain sperm in men with non-obstructive azoospermia [39].
4. Results Most series have indicated that there are no individual characteristics of the men themselves that will further predict the chance of achieving pregnancy after a sperm retrieval
procedure. There does appear to be a difference between results for obstructive and non-obstructive azoospermia, partly because not all men with non-obstructive azoospermia have retrievable sperm. 4.1. Obstructive azoospermia Pregnancy rates after sperm retrieval will depend on the certainty with which the diagnosis of obstruction has been made prior to retrieval as well as on the overall results achieved at that IVF unit. Belker et al. [40] reported a clinical pregnancy rate of 40% when testicular sperm retrieval was performed using percutaneous fine-needle aspiration, whereas Gil-Salom et al. [41] described a 27% pregnancy rate with open testicular biopsy. 4.2. Non-obstructive azoospermia The results of sperm retrieval by open testicular biopsy for men with non-obstructive azoospermia are made up of two different components. The first is the ability to retrieve sperm from the testis, the second is the pregnancy rate after ICSI using those sperm. One of the factors that may reduce the chance of sperm retrieval is repeating the testicular biopsy within 6 months; this also increases the risk of permanent testicular devascularization [5]. Reports of the chance of retrieving sperm have been as high as 90% for men without obstruction in a small series and as low as 50% in larger series. Obviously, the more carefully the men are selected before retrieval, the better is the likelihood of finding sperm with TESE. One team initially reported that 80% of men had sperm retrieved with TESE. The fertilization rate per injected oocyte was 48%, with an overall clinical pregnancy rate of 20% per attempt at TESE [3]. Subsequent results from Belgium suggest that the rate of sperm retrieval is closer to 50% per TESE procedure [4]. Another group reported that 10 out of 16 (62%) men without obstruction had sperm extracted with TESE. The fertilization rate per injected oocyte was 58%, and 5 out of 10 (50%) of those undergoing ICSI achieved a clinical pregnancy rate of 31% per attempt at TESE [42].
5. Summary Testicular sperm retrieval from men with azoospermia is now possible with acceptable-to-excellent pregnancy rates when used alongside ICSI. Advances in both sperm retrieval and assisted reproduction have widened the potential of fertility treatment when the only management options a few years previously had been donor insemination and adoption. Extensive multiple biopsies from every area of the testis are often performed in an effort to find sufficient spermatozoa for ICSI. This can result in a great deal of testicular damage and an alteration in testicular function, including testicular atrophy. This damage can be minimized by using
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needle rather than open biopsy to obtain spermatozoa for ICSI. This is possible and has proved to be successful in cases of obstructive azoospermia; however, when spermatogenesis is deficient, needle biopsy is much less likely than open biopsy to find the rare foci of spermatogenesis necessary for ICSI. Since our ultimate goal is to reduce the amount of testicular tissue removed, it has been noted that the scarce foci of normal spermatogenesis in cases of non-obstructive azoospermia could be detected directly under the operating microscope. Only tiny portions of testicle would then need to be removed using this microsurgical technique. Another advance is the ability to use cryopreserved testicular spermatozoa, which will limit the number of sperm retrieval procedures necessary to achieve fertility for a couple. Specific genetic abnormalities are associated with both types of azoospermia, and a careful evaluation of the cause of azoospermia and genetic counselling are indicated for all azoospermic men. A significant proportion of non-obstructive azoospermic men will fail to have sperm retrieved with testicular sperm extraction procedures. To date, there has been no good prognostic indicator of sperm retrieval success in such cases. The most sensitive markers are currently fine-needle aspiration mapping and open testicular biopsy. The search should continue to find more reliable indicators. The choice of a suitable testicular sperm retrieval technique will depend on the aetiology of the azoospermia, the therapeutic purpose of the procedure, the expertise of the operator and the available facilities. Acknowledgements The authors thank Dr. Dee Kini FRCOG, the director of the IVF unit at the Al-Mana General Hospital, Dammam, for his assistance in the preparation of this manuscript. References [1] Steptoe PC, Edwards RG. Birth after the re-implantation of a human embryo. Lancet 1978;2:366. [2] Palermo GD, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992;340:17. [3] Devroey P, Liu J, Nagy Z, et al. Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermia. Hum Reprod 1995;10:1457. [4] Kahraman S, Ozgur S, Alatas C, et al. Fertility with testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermic men. Hum Reprod 1996;11:756. [5] Schlegel PN, Palermo GD, Goldstein M, et al. Testicular sperm extraction with intracytoplasmic sperm injection for nonobstructive azoospermia. Urology 1997;49:435. [6] Girardi SK, Schlegel PN. Microsurgical epididymal sperm aspiration: review of techniques, preoperative considerations, and results. J Androl 1996;1:5. [7] Reijo R, Lee T, Salo P, et al. Diverse spermatogenic defect in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nat Genet 1995;10:383.
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[35] Ben-Yosef D, Yogev L, Hauser R, et al. Testicular sperm retrieval and cryopreservation prior to initiating ovarian stimulation as the first line approach in patients with non-obstructive azoospermia. Hum Reprod 1999;14:1794. [36] Manning M, Junemann KP, Alken P. Decrease in testostrone blood concentrations after testicular sperm extraction for intracytoplasmic sperm injection in azoospermic men. Lancet 1998;352:37. [37] Jarow JP. Clinical significance of intratesticular arterial anatomy. J Urol 1991;145:777. [38] Harrington TG, Schauer D, Gilbert BR. Percutaneous testis biopsy: an alternative to open testicular biopsy in evaluation of the subfertile man. J Urol 1996;156:1647. [39] Schlegel PN. Testicular sperm extraction: microdissection improves sperm yield with minimal tissue excision. Hum Reprod 1999;14: 131. [40] Belker AM, Sherins RJ, Coulan CB, et al. High fertilization and pregnancy rates obtained by nonsurgical percutaneous needle aspiration of testicular sperm. J Urol 1996;155:364. [41] Gil-Salom M, Minguez Y, Rubio C, et al. Intracytoplasmic testicular sperm injection: an effective treatment for otherwise intractable obstructive azoospermia. J Urol 1995;154:2974. [42] Schlegel PN, Palermo GD, Goldstein M, et al. Testicular sperm extraction with ICSI for nonobstructive azoospermia. Urology 1997;49(3):435–40.