Innovations in surgical management of nonobstructive azoospermia.

Testicular sperm extraction (TESE) technique and intra-cytoplasmic sperm injection are breakthrough fertility treatments for men with nonobstructive azoospermia (NOA). Newer advances such as the microdissection-TESE (micro-TESE) technique have continued to build upon past success by improving sperm retrieval and minimizing the postoperative complications compared to TESE. However, even with micro-TESE, sperm retrieval success has ranged from 40% to 60% due to the technique's dependence on surgeon and embryologist experience. While postoperative complications are minimal relative to the traditional TESE technique, testicular tissue must still be extracted without the knowledge of whether sperm are present in biopsies. In this review, we discuss the innovations in the surgical management of men with NOA and describe the novel experimental approaches that can improve sperm retrieval success.

INTRODUCTION

Nonobstructive azoospermia (NOA) or azoospermia secondary to testicular failure affects approximately 10% of infertile men. The underlying histology of NOA can be sertoli cell-only, maturation arrest, or hypospermatogenesis. Sperm retrieval techniques for men with NOA requires a procedure such as testicular sperm extraction (TESE) or the microdissection-TESE (micro-TESE) followed by intra-cytoplasmic sperm injection (ICSI).[1] Since 1993, TESE was used to successfully retrieve sperm followed by in vitro fertilization (IVF) with ICSI, where only a single sperm is injected into the egg for fertilization.[2] TESE and ICSI provided the first opportunity for men with NOA to father biological children. In 1999, Schlegel showed that sperm retrieval success could be improved from ~40% to ~60% using an operating microscope to identify potential sperm containing seminiferous tubules in a technique known as micro-TESE.[3] While the introduction of the microdissection technique has shown improved success in sperm extraction, there remains much room for improvement in safety, efficacy, and efficiency in sperm retrieval for men with NOA. In this review, we will discuss some of the latest advances on the horizon that have demonstrated the potential to improve sperm extraction success while minimizing the amount of tissue removed and duration of surgery [Table 1].

Table 1

A comparison of current and experimental surgical techniques and modalities in the management of nonobstructive azoospermia

TESTICULAR SPERM EXTRACTION/INTRA-CYTOPLASMIC SPERM INJECTION

TESE for use in IVF with ICSI was first reported in 1993 by Schoysman et al.[2] TESE enabled urologists to extract sperm from men who lacked sperm in the ejaculate but had viable sperm in the seminiferous tubules. TESE is performed with a small incision in the tunica albuginea, allowing the surgeon to extract seminiferous tubules. Several tubules are collected and processed for sperm. For men with NOA, TESE provides approximately a 20–45% chance of successful retrieval and can often be done without general anesthesia in the clinic.[3] While convenient and straightforward, TESE removes large sections of the seminiferous tubules and can cause inflammation and potential rupture of the testicular vasculature.[4]

Since TESE collects relatively few sperm, it is nearly always accompanied by assisted reproductive techniques. The most important of these techniques is IVF with ICSI. ICSI, first described in 1992 by Palermo et al., is performed when a single sperm is injected into an egg instead of simply placing sperm in a solution with the egg as performed in traditional IVF.[15] The fertilized egg is then placed into the woman's uterus for development. Only a few sperm are necessary for fertilization with ICSI, which enabled techniques such as TESE to be performed for sperm retrieval in the fertility treatment of men with NOA.

MICRO-TESTICULAR SPERM EXTRACTION

Micro-TESE was first reported in 1999 by Schlegel.[3] Using an operating microscope, the surgeon is able to better visualize regions containing spermatozoa by identifying differences in the size and opacity of seminiferous tubules. Initially, the testes are visualized under 6–8x[316] magnification to allow optimal visualization of the blood vessels and a single wide incision in an avascular region of the tunica albuginea is made to expose underlying testicular parenchyma [Figure 1]. Next, under higher magnification (15–25x), the surgeon identifies potential spermatozoa containing tubules and dissects them. Tubules with spermatozoa will appear to be larger, thicker, and more opaque, although this may not be the case in all circumstances since sperm identification is possible only after tubules are biopsied and mechanically processed. The sample is then cut, mechanically processed, and examined by a skilled embryologist for viable spermatozoa. In an initial nonrandomized prospective study, spermatozoa retrieval improved from 45% using TESE to 63% using the microdissection technique.[3] A larger retrospective clinical study with 543 TESE procedures showed similar results with 57% overall spermatozoa retrieval success with the microdissection technique versus 32% with traditional TESE.[4]

Figure 1

Microdissection testicular sperm extraction involves wide incision of tunica albuginea to allow extensive visualization of testicular tubules. Permission for reproduction obtained from Elsevier Publishing[4]

While micro-TESE dramatically increases spermatozoa retrieval success, the operation remains a technically challenging one that demands a longer operating time.[1718] In a retrospective clinical analysis by Ramasamy et al. of 793 micro-TESE attempts, mean operative duration ranged from 1.8 to 2.7 h with longer times associated with decreased spermatozoa retrieval success (from 89% to 30% after 2 h).[19] In a retrospective clinical analysis by Ishikawa et al., the group showed that in a single urologist performing micro-TESE on 150 men with NOA, sperm retrieval increased from 32% in the first 50 cases to 48% in the last 50 cases with a learning curve of about 50 cases with operating times decreasing from 114 ± 32 min in the first 50 cases to 85 ± 18 min in the last 50 cases.[17] In addition to an experienced microsurgeon, successful spermatozoa retrieval depends on an expert embryologist who is well-versed in processing and analyzing testicular tissues samples for sperm.[7]

In special population, such as men with severe testicular atrophy (testicular volume ≤2 mL) as seen in many patients with Klinefelter's syndrome, smaller testicular volume have not been shown to be a predictor of decreased success with micro-TESE. In a retrospective analysis by Bryson et al. 1127 patients with NOA who underwent micro-TESE with ICSI, sperm retrieval rates for patients with testicular volumes of ≤2 mL, 2–10 mL, and >10 mL were 54.7%, 56.25, and 55.1%, P = 0.53, respectively.[16] Of the 106 patients with testicular volumes of ≤ 2 mL, 82.2% had Klinefelter syndrome. Though this study encompasses information from a confined population, patients with conditions of severe testicular atrophy such as Klinefelter's should not be discouraged from undergoing the micro-TESE procedure for sperm extraction.

MULTIPHOTON MICROSCOPY

A challenge faced by micro-TESE use in men with NOA has been the difficulty of successfully differentiating between seminiferous tubules with normal and abnormal spermatogenesis.[8] A technique that can potentially identify the presence of sperm within the tubules without the need for extraction of testicular tissue is multiphoton microscopy (MPM). MPM uses a low energy infrared femtosecond pulse laser with two- and three-photons to produce the excitation of intrinsic molecules causing autofluorescence from intrinsic fluorophores combined with second harmonic generation by supramolecular structures to produce detailed images of underlying tissue.[820] The tissue can be optically sectioned allowing for real-time high-resolution images without the conventional use of physical section, labeling, or staining.[8] In one of the earliest studies using MPM to evaluate spermatogenesis in seminiferous tubules, Ramasamy et al. showed the ability of MPM to clearly distinguish the underlying microstructure of the seminiferous tubules in a mouse model to allow for sperm detection and histological characterization of individual tubules [Figure 2].[7] In a follow-up pilot study by Najari et al., the MPM technique was used in seven men with normal or abnormal spermatogenesis to test the ability of MPM to characterize seminiferous tubules compared to those obtained from hematoxylin and Eosin (H and E) staining.[8] The study showed a 92% concordance rate of diagnosis between the two techniques in men with NOA while showing a concordance rate for all men with normal to be 78%. The study also showed the similar proportions of the categorizations of seminiferous tubules with abnormal versus normal spermatogenesis.

Figure 2

Germ cell depleted, sertoli cell-only seminiferous tubule (a) and tubules with spermatogenesis (b). Low-power multiphoton microscopy (A). Reduced from ×4. High-power multiphoton microscopy (B). Reduced from ×25. H and E, reduced from ×25 (C). Permission for reproduction obtained from Elsevier Publishing[7]

Though the studies on MPM are still in their preliminary phases, the MPM technique has shown promising results that can enhance the ability of the surgeon to improve sperm retrieval by correctly identifying seminiferous tubules with sperm and potentially decreasing the operative time in the process. While it only uses a low energy laser, the MPM laser-guided technique will still need to be evaluated for its long-term safety in future prospective studies as lasers can potentially induce genetic abnormalities in gametes used for IVF with ICSI.

FULL FIELD OPTICAL COHERENCE TOMOGRAPHY

Full-field optical coherence tomography (FFOCT) is a technique that uses the principle of white light interference microscopy to produce the high-resolution images of unprocessed and unstained excised tissue.[10] In one of the first investigations of the use of commercial prototype use of FFOCT, called Light-CT™, on the quality of histological samples of various rat organs, Jain et al. was able to successfully visualize the normal histology of nine different rat organs, including the testes.[10] The images produced by FFOCT allows for the clear recognition of seminiferous tubules, the layers of germ cells that line the tubule, and the distinct tubule lumen with intra-luminal sperm. In one of the first studies of using FFOCT to evaluate spermatogenesis in the seminiferous tubules in a mouse model, Ramasamy et al. demonstrated that FFOCT was able to successfully image the seminiferous tubules and determine the status of spermatogenesis.[11] In the pilot animal study, normal rat testes were compared with those that have been injected with two doses of busulfan – an alkylating agent that temporarily arrests spermatogenesis. The FFOCT technique showed seminiferous tubules of relatively uniform size and shape (diameter 328 ± 11 μm) and hair-like structures the indicative of spermatogenesis in normal rat testis samples compared to the heterogeneity in shape and the size of tubules (diameter 178 ± 35 μm) with only 10% of tubules showing normal spermatogenesis in the mice treated with busulfan. The FFOCT findings correlated well with the H and E staining of the same specimens [Figure 3].

Figure 3

Comparative full field optical coherence tomography and H and E-stained histology (a) Testis of a normal rat shows seminiferous tubules with relatively uniform size and shape (b) H and E histology stain of the same specimen. Arrows point to the sperm within the tubule lumen. (c) Seminiferous tubules in the testis of a rat treated with busulfan, showing thinner tubules and a greater degree of heterogeneity in size and shape with ~10% normal spermatogenesis. (d) H and E staining of the same specimen. Field of view in each panel: 1 mm2 Permission granted under the creative commons attribution license[11]

The FFOCT technique holds much promise in improving the success of sperm extraction with its potential for reducing the number of biopsies performed and decreasing the operative time for the micro-TESE procedure. The technique allows for rapid imaging of fresh tissue without the need for extrinsic labeling agents. With the halogen lamp as the illuminating power source, the technique offers a relatively safer option compared to laser imaging techniques such as MPM, making it ideal for ICSI as it decreases the chances of photo-damage and mutations in extracted sperm.[11] In addition, the FFOCT technique, compared to traditional confocal microscopy, is much faster (one frame/s) in its generation of images and can cover a relatively larger area of tissue. The major limitations for this technique are the lack of ability to image cellular and nuclear details, its limited depth of imaging, and the constraint of the prototypes to image ex vivo specimens only.[1011] Despite the positive findings in the studies mentioned, FFOCT will still need to be tested in human tissue and further evaluated on its safety and efficacy.

RAMAN SPECTROSCOPY

Raman spectroscopy (RS) is a nondestructive, label-free technique that utilizes laser-based optics to determine the biochemical structures of living tissue. RS uses a low-power, monochromatic laser to excite molecules. Each molecule will in turn uniquely scatter these photons, creating a spectrum specific to each molecular structure. With this technique, investigators can differentiate tissues to generate a “fingerprint” spectrum for each type of tissue. RS is used clinically to identify tumors, analyze serum for oral cancer makers, and detect DNA damage in human sperm.[212223] In a 2014 study, Osterberg et al. demonstrated that this technique can be used to differentiate between tubules with sertoli cell-only histology and tubules with spermatogenesis [Figure 4]. This technique was 96% sensitive and 100% specific in distinguishing the presence of spermatogenesis in the seminiferous tubules of rat models.[13] In another study reported in 2014, Liu et al. demonstrated the ability of RS to distinguish between NOA and obstructive azoospermia with 90% sensitivity and 85.71% specificity.[12] However, this study was limited in that testicular tissues of men with normal spermatogenesis were not included as controls.

Figure 4

(a) Mean processed spectra for sertoli cell-only (red curve) and active spermatogenesis (blue curve) with 1000 and 1690 cm−1 discriminatory Raman peak intensity, respectively. (b) Representative testicular biopsy shows active spermatogenesis. (c) Representative testicular biopsy shows sertoli cell-only. (b and c), H and E, reduced from ×200. Permission for reproduction obtained from Elsevier Publishing[13]

The specificity and sensitivity of RS are greater than any other technique discussed in this review. Further, this technique is nondestructive and the sperm remains viable for ICSI. Conversely, while this technique is touted as nondestructive, the overall safety of RS use on humans has not been thoroughly investigated.[12] Unfortunately, RS is not an imaging modality; instead, the surgeon uses a flexible probe to analyze testicular tissue using an algorithm to decide whether or not to extract tubules. Each analysis currently takes approximately 100 s (~2 min) – thus, while this is a viable real-time analytical tool, it can be inefficient compared to some of the other techniques.[13] Finally, since RS is a light-based spectroscopy, careful fine tuning is required, and excess background lighting can cause errant results, diminishing the reliability of this technique.[13]

SPERM ANTIBODY DETECTION

The final technique reviewed utilizes fluorescent microscopy to identify antibody-labeled sperm in seminiferous tubules during micro-TESE procedures. Greenhalgh et al. first described this technique in 2009 using monoclonal antibodies and a traditional fluorescent microscope.[24] Smith et al. improved on this concept in 2012 using a fiber optic confocal fluorescent microscope which has a flexible probe and can be used during surgery to produce in vivo cellular images.[14] This technique requires an injection of sperm-specific monoclonal antibodies into the rete testis prior to surgery. Once the testicle has been opened during the micro-TESE procedure, an image of the tubules is produced in < 1 s as the fluorescently labeled sperm are mapped out in the image – thus allowing the surgeon to quickly identify and collect the sperm-filled tubules.

Both studies were based on a mouse-model and have not been validated in human specimens. While the generation of an in vivo cellular image would decrease surgical time as well as decrease testicular damage, it remains unclear whether the antibody-bound sperm is viable for assisted reproductive techniques. Further, it is uncertain what the long-term effects are of fluorescent-tagged monoclonal antibodies in humans. Finally, while the monoclonal antibodies bound well to mouse sperm in both studies, it is unlikely that monoclonal antibodies would be suitable for the myriad of morphological sperm found in the human population, thus limiting the effectiveness of this technique.

CONCLUSION

There is a need for improved ability to identify sperm containing regions within the testis of men with NOA. Currently, skilled surgeons using a microscope during TESE have shown improved sperm retrieval success compared to conventional TESE. Nevertheless, challenges such as long operative duration and the necessity to remove tissue to positively identify sperm limits micro-TESE's wide application. Therefore, it is imperative that we develop a technique using innovative modalities that can achieve similar success but at the same time overcome some of the current challenges. While the safety of the techniques discussed within this review has yet to be fully investigated, we expect future studies to benefit the surgical treatment of men with NOA.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.