Historical perspective of surgical sperm retrieval techniques for nonobstructive azoospermia

The advent of intracytoplasmic sperm injection, along with the realization that many men with azoospermia due to primary testicular failure may have a few spermatozoa in their testes, has resulted in the revolutionary possibility of azoospermic men fathering their own genetic offspring. Initially, random sampling of the testes by testicular aspirations or multiple testicular biopsies was used to retrieve sperm in these men. However, these blind techniques could miss small foci of sperm. The development of microdissection testicular sperm extraction (micro-TESE) facilitated visual inspection of the seminiferous tubules under microsurgical magnification to allow for the selection of the more dilated tubules that were likely to contain sperm. This paper reviews the historical progression of surgical sperm retrieval techniques in men with nonobstructive azoospermia (NOA). Until the 1990s, the only options available for couples with male partners with NOA were adoption or use of donor sperm. Three events dramatically changed this landscape. The first one was the development of intracytoplasmic sperm injection (ICSI) which enabled the achievement of a viable pregnancy with extremely small numbers of spermatozoa.1 Then, it was found that sperm retrieved from the testicle may be used successfully for in vitro fertilization (IVF) and ICSI.2,3 Finally, the recognition came that even in men with severe testicular failure, a few foci of spermatogenesis may be present, which could be retrieved surgically4 and used for ICSI to produce a live, healthy offspring. This resulted in an explosion of surgical techniques to retrieve sperm in men with NOA. This article presents a historical overview of these developments. Simultaneously, this has resulted in other developments. Research studies have explored predictors of sperm retrieval, clinicians have attempted to improve sperm retrieval by giving hormonal therapy prior to sperm retrieval, and embryologists have developed techniques for recovery and cryopreservation of rare sperm obtained from the testicular tissue. SURGICAL SPERM RETRIEVAL Innovative attempts at surgical retrieval of sperm have a long history. In 1955, Hanley,5 in his Hunterian Lecture at the Royal College of Surgeons, described tucking amniotic tissue, loosely folded into a ball, under the tunica of the epididymis to create an artificial spermatocele to recover sperm. Subsequently, there were attempts to use alloplastic spermatoceles to retrieve sperm, but even though sperm were retrieved, pregnancies were elusive.6 The first live births after insemination with these sperm were reported by Cruz7 and Kelâmi.8 However, the use of epididymal sperm for cervical or intrauterine insemination was only occasionally successful and it was only after the advent of IVF, and especially ICSI, that high fertilization and pregnancy rates could be achieved. The first success of IVF with epididymal sperm was reported in 1985 in a man who had twice failed vasectomy reversal.9 Soon after, Silber et al.10 published their technique of microsurgical epididymal sperm aspiration (MESA) for use in men with aplasia of the vasa defentia. Later, Tsirigotis et al.11 simplified this into a percutaneous technique for aspirating epididymal sperm (PESA). However, in some cases of obstructive azoospermia (OA), no sperm could be retrieved from the epididymis, and this led researchers to consider the use of testicular sperm. In 1993, Craft et al.2 demonstrated that testicular sperm could fertilize eggs during IVF, and Schoysman et al.3 achieved a live birth with ICSI. The fertilizing potential of testicular sperm from men with OA was confirmed in a larger study,12 and equal success was reported even with cryopreserved testicular sperm.13 For these men with OA, adequate sperm could easily be obtained through a conventional open biopsy of the testis. These successes led to an exploration of the possibility of finding sperm in the testes of men with testicular failure and the use of these sperm for ICSI. In 1995, using conventional random biopsies, Devroey et al.4 were successful in finding sperm in 13 of 15 men with NOA and reported a 47.8% fertilization rate with three pregnancies. In the same year, other clinicians also reported finding sperm in men with testicular atrophy,14 or with Sertoli cell-only syndrome (SCOS) and high follicle-stimulating hormone (FSH) levels.15 However, these cases required multiple biopsies to recover sufficient sperm. One study reported 2.8 ± 2.5 (mean ± standard deviation [s.d.]) biopsies in men with SCOS and 4.5 ± 4.2 (mean ± s.d.) biopsies when maturation arrest was present.16 Hence, a variety of alternative surgical approaches were developed to recover sperm from the testes of men with NOA. Fine-needle aspiration (FNA) of the testis is a well-established diagnostic procedure.17 Craft and Tsirigotis18 reported using this technique to retrieve sperm for ICSI in men with OA. Lewin et al.19 suggested that this could also work in cases with NOA. Their technique of testicular fine-needle aspiration (TEFNA) involved 15 punctures of the testis with a 23-gauge needle attached to a 20-ml syringe with an aspiration handle. Multiple passes were made in various directions while applying suction. This resulted in macerated tissue and fluid being aspirated into the syringe. This aspirate was checked for sperm. They reported finding sperm in 48% of men with SCOS, 46% of men with maturation arrest, and 66% of men with nonmosaic Klinefelter syndrome.19 Since TEFNA is technically easy, it became a popular procedure. However, subsequent studies questioned the efficacy of TEFNA (or testicular sperm aspiration [TESA], as it was also called). Friedler et al.20 compared TEFNA (6 punctures) with open biopsies (3 biopsies on each side) in the same patients and found 11% successful sperm retrievals in TEFNA versus 43% with open biopsy. Tournaye et al.21 reported that even in OA cases, TESA failed to retrieve sperm in 2 of 53 men. In another modification, several studies suggested that sperm retrieval rates by TESA could be improved using a color Doppler to identify testicular regions with more arterial perfusion.22–24 TEFNA was subsequently modified with the use of larger needles. These allowed percutaneous aspiration of multiple seminiferous tubules equivalent to an open testicular biopsy. Morey et al.25 used a biopsy gun to extract a core of testicular parenchyma. Mallidis and Baker26 recommended the modified Menghini and Turner biopsy needles after comparing a variety of needles. Ezeh et al.27 and Shah28 aspirated a core of testicular tissue by applying suction with a 19- or 18-gauge butterfly needle, respectively. Shah29 also described the single seminiferous tubule (SST) technique as an open method of obtaining multiple testicular biopsies with minimal trauma. The scrotum was incised to expose the testis and the tunica albuginae was punctured with a 20G needle. Pressure was applied on the testis resulting in the protrusion of a seminiferous tubule through the puncture hole. This was grasped with a non-serrated micro-forceps and pulled out of the testis till it snapped or till a long length of tubule was pulled out. The micro-biopsy thus obtained was sent to the IVF laboratory to check for sperm. If negative, the procedure was repeated at multiple locations till the entire testicular surface was mapped. A major change from all these techniques was the introduction of micro-TESE by Schlegel30 in 1999. In 1997, Schlegel and Su31 raised concerns that the multiple and large conventional biopsies used to find sperm in NOA men were causing significant damage to the testes. Instead, Schlegel30 suggested an approach in which the testis was exposed and bivalved, and the parenchyma was then explored under an operating microscope. The magnification allowed the identification of tubules that were larger than the surrounding flat and thin seminiferous tubules. These larger tubules were thought to be the ones with spermatogenesis, and only these were extracted and sent to the IVF laboratory for sperm extraction. In their initial report, Schlegel and Su31 performed both conventional biopsies and micro-TESE on 27 men and compared sperm retrieval rates. Sperm was found in 11 (41%) men through conventional biopsy and micro-TESE. In an additional 6 (22%) men, sperm were found only by micro-TESE, highlighting the additional benefit of micro-TESE. In one case, however, sperm were found only in the conventional biopsies but not in the micro-TESE samples. This can happen when the testicular parenchymal pattern is uniform and there is no visible distinction between the tubules. In such a situation, finding a sperm-bearing tubule is just a matter of chance and micro-TESE does not confer any advantage. Many studies have confirmed that micro-TESE is the most efficient method for finding sperm in men with NOA,32 but its advantage is primarily in cases with SCOS or testicular atrophy.33,34 Though micro-TESE was conceived as a technique to minimize testicular damage by extracting only small amounts of tissue, subsequent studies have shown that it is not innocuous and a significant proportion of men undergoing micro-TESE develop hypogonadism, usually temporary, but occasionally permanent.35,36 Hence, alternative approaches have been proposed to allow for the best chance of sperm retrieval while keeping testicular damage to a minimum. Several authors37,38 have suggested the use of different sperm retrieval techniques in the same surgical session, starting with less invasive methods and then progressing to more extensive procedures until sperm are retrieved. Shah37 describes a single-session, multi-stage sperm retrieval procedure that starts with 3 to 4 percutaneous needle biopsies from one testis. If no sperm are found on the first side then the needle biopsies are performed on the opposite testis. If no sperm are seen in the needle biopsies, then the scrotum is incised and one testis is exposed. Multiple samples of the seminiferous tubules are taken from the surface of the testis using the atraumatic SST technique described earlier. If the extracted tubules are thin and gelatinous, then only a few samples are taken. If the tubules appear healthy, then 20 to 30 micro-biopsies are taken from all over the testis and are sent to the IVF laboratory to check for sperm. If negative, the same procedure is repeated on the other testis. If sperm are still not found, one testis is bivalved and a comprehensive micro-TESE is performed. If the first side is negative, the micro-TESE is repeated on the contralateral side. This approach avoids more invasive procedures in cases where sperm can be found by simpler methods and may reduce the costs, time, and efforts involved in surgery, though this has not been validated through comparative studies. Another approach is based on the concept of diagnostic mapping of testicular spermatogenesis through FNA at multiple sites from each testis.39,40 The aspirate is air-dried, stained and examined at a later date for the presence of sperm. If sperm are found then ICSI with fresh sperm retrieval is planned. The number of sperm present/absent at each aspiration site is used to guide which sperm retrieval technique should be used (aspiration or micro-TESE), and which site of the testis should be explored. This approach has also been used to identify loci of sperm in cases of previously failed micro-TESE.41 RECENT DEVELOPMENTS Although micro-TESE is considered the gold standard technique for surgical sperm retrieval, it is an aggressive technique that still fails to retrieve sperm in half of the cases. Hence, in parallel with the developments in surgical techniques, other studies have focused on how sperm retrieval may be made more predictable43 and precise and whether treatment before sperm retrieval may improve recovery rates. Many studies assessed the utility of hormone tests to estimate the chances of sperm retrieval, but the results have been conflicting. While some found lower FSH to be predictive of better chances of sperm retrieval,42,43 other studies reported that hormone levels did not predict sperm retrieval.44 Some studies have used seminal biomarkers to predict spermatogenesis in men with NOA with variable results.45,46 Magnetic resonance imaging has also been used to predict the presence of spermatogenesis.47 Several studies evaluated the possible role of hormonal therapy prior to micro-TESE. However, these studies were small, and the evidence is limited. A recent meta-analysis suggested that hormone therapy may be valuable in men with eugonadotropic testicular failure but not in those who were hypergonadotropic.48 Researchers have also looked at intraoperative aids to identify sperm-bearing seminiferous tubules, but none has been found useful or practical enough to be incorporated into standard practice. Technologies used have included multiphoton microscopy,49 full-field optical coherent tomography,50 and tissue perfusion monitoring using narrow band imaging.51 CONCLUSIONS Retrieving sperm from the testes of men with testicular failure, and using these sperm by ICSI to produce a viable pregnancy, is one of the major advances in the treatment of severe male infertility. This article presents a historical overview of the development of various techniques for the retrieval of sperm from the testes of men with NOA, and highlights future areas of research. AUTHOR CONTRIBUTIONS RS contributed to the conceptualization. All authors wrote and edited the article, and read and approved the final manuscript. COMPETING INTERESTS All authors declare no competing interests.