Abstract
The development of surgical sperm retrieval procedures can be considered as the single most important breakthrough in the field of male infertility. Various testicular sperm retrieval procedures exist and are indicated in patients with obstructive and nonobstructive azoospermia, as well as patients with high levels of sperm DNA fragmentation and severe derangements in semen parameters. Microsurgical testicular sperm extraction can be considered the gold standard retrieval method as it allows meticulous and selective sampling of sperm-containing seminiferous tubules, yielding the highest retrieval rate in comparison to other surgical sperm retrieval methods.
6.1 Introduction
Surgical sperm retrieval can be considered the single most important breakthrough that the field of male infertility has witnessed. The advancements in assisted reproductive technology (ART) in the 1970s allowed physicians to investigate alternative sources of sperm from men with azoospermia. This clinical condition, defined as the absence of sperm in the ejaculate, is prevalent in about 1 percent of all men and in up to 15 percent of men seeking conception [1]. Nonobstructive azoospermia (NOA) is perhaps the most severe form of male infertility; in the past, men with this condition were considered sterile and adoption or assisted reproduction using donated sperm were the only available options for the couple to become parents. However, in recent years and with the introduction of sperm retrieval techniques, conception was made possible using sperm extracted from the testes of men with NOA. This realization came into action in 1994 after the work of Devroey et al. [2], who retrieved sperm via conventional testicular sperm extraction (cTESE), reporting three pregnancies with intracytoplasmic sperm injection (ICSI). In 1995, Craft and Tsirigotis [3] proposed testicular sperm aspiration (TESA) as a simplified method of sperm recovery for men with NOA. In 1998, and following the reports of a few studies demonstrating the detrimental effects of multiple cTESEs on testicular function [4,5], Schlegel and Li [6] introduced the microsurgical testicular sperm extraction (micro-TESE) method, which allowed the performance of magnified selective biopsies of promising seminiferous tubules, thereby minimizing testicular damage. Finally, in 2001, Shah [7] introduced the single seminiferous tubule (SST) biopsy as a less traumatic alternative method allowing multiple biopsies through puncture holes in the tunica albuginea. This chapter is aimed at describing the indications of testicular sperm retrieval, the surgical techniques, and their clinical outcomes.
6.2 Indications for Testicular Sperm Retrieval
Testicular sperm retrieval can be performed on patients with obstructive azoospermia when attempts at re-canalizing the reproductive tract have failed or are not possible (Table 6.1) [8]. While the retrieval of sperm from the epididymis is favored in such cases, it may fail especially when intratesticular obstruction is present. On the other hand, testicular sperm retrieval is commonly performed for men with NOA in search of focal areas of spermatogenesis. In about 30–60 percent of cases, such areas can be identified, resulting in successful sperm retrieval [9]. Throughout the years, the indications of testicular sperm retrieval have extended to cover conditions other than azoospermia. These include (1) the presence of abnormally high levels of sperm DNA fragmentation; and (2) cryptozoospermia, which is the presence of very few sperm (often with poor quality) in the ejaculate. Sperm DNA fragmentation has been investigated in recent years and the reports have indicated that it plays a crucial role in fertilization, early embryo development, and pregnancy rate [10–14]. It is now believed that high levels of sperm DNA fragmentation could alter the outcome of ART by raising the failure and miscarriage rates following these procedures [15,16]. While sperm DNA fragmentation can occur secondary to defects in chromatin compaction during spermiogenesis, the majority of cases are believed to arise from oxidative stress-related insults during sperm passage through the male reproductive tract [17–21]. This finding has been confirmed by several studies reporting higher levels of sperm DNA fragmentation in the ejaculates of non-azoospermic men, compared with their intratesticular sperm samples [22–24]. A systemic review by Esteves et al. reported a mean difference in sperm DNA fragmentation rate of –24.6 percent between ejaculate and intratesticular sperm samples [25]. A number of studies have compared the outcome of ICSI using testicular sperm versus ejaculated sperm [22,26,27]. Chapter 8 covers the summary evidence, indications, and protocol concerning testicular sperm retrieval for non-azoospermic men.
Table 6.1 Indications of testicular sperm retrieval
| Nonobstructive azoospermia |
| Obstructive azoospermia |
| High sperm DNA fragmentation |
| Cryptozoospermia |
Patients with cryptozoospermia ejaculate very few sperm of low motility that may reduce the success rate of ICSI [28]. Therefore, a number of studies have investigated whether testicular sperm retrieval in cases of cryptozoospermia may be associated with improvements in ICSI outcome [28–30]. A recent meta-analysis investigated 761 ICSI cycles performed on men with cryptozoospermia: 541 ICSI cycles with ejaculated sperm, 153 ICSI cycles with fresh testicular sperm, and 67 ICSI cycles with frozen–thawed testicular sperm. The results revealed that testicular sperm did not significantly improve the fertilization rate in comparison to ejaculated sperm (relative risk = 1.08 with 95 percent confidence interval (CI) 0.98–1.32; p = 0.12); however, a significant improvement in good-quality embryo rate (1.17, 95 percent CI 1.05–1.30, p = 0.005), implantation rate (95 percent CI 1.02–2.26, p = 0.04), and pregnancy rate (1.74, 95 percent CI 1.20–2.52, p = 0.004) were observed with testicular sperm in comparison to ejaculated sperm in patients with cryptozoospermia [30].
6.3 Surgical Sperm Retrieval
6.3.1 Preoperative Care
Patients should be fully informed about the intended surgical procedure, its potential complications, and the expected outcome before signing the informed surgical consent. Their medical history should be reviewed and optimized before undergoing surgery, especially for patients with diabetes mellitus, cardiac conditions, or bleeding disorders. Antiplatelets, anticoagulants, and nonsteroidal anti-inflammatory medications should preferably be stopped one week before the procedure. Shaving of the scrotal wall hair should be advised, especially when open surgery is intended. Prophylactic antibiotics (e.g., first-generation cephalosporin) are required, particularly for open procedures.
6.3.2 Anesthesia
Testicular sperm retrieval is a relatively simple procedure that is performed in the operating theater under general or spinal anesthesia. Alternatively, spermatic cord block using lidocaine 2 percent with/without intravenous sedation can be performed in the outpatient setting, especially when percutaneous aspiration is intended.
6.3.3 Methods for Surgical Sperm Retrieval (Surgical Protocols)
6.3.3.1 Testicular Sperm Aspiration
Various modifications for TESA have been described. A simple aspiration cytology procedure can be performed using a 22 G butterfly needle, which is inserted into the testicular tissue while applying suction with a 20 ml syringe [3]. The aspirated fluid is then checked for sperm. Alternatively, needle aspiration biopsy (NAB) can be performed using an 18 G intravenous cannula inserted perpendicularly in the anterior surface of the testicle [7]. Once the cannula punctures the tunica albuginea, the needle is retrieved and the cannula is rotated 180 degrees a number of times. The cannula is then connected to a 20 ml syringe through an IV extension tube. While applying suction, the cannula is cautiously pulled and pushed within the testis until a column of testicular tissue can be visualized. The tubing is then clamped while under suction and the cannula is slowly pulled out of the testis. As the cannula emerges from the scrotal skin, a loop of seminiferous tubule will be observed, which can be grasped with microsurgical forceps, pulling additional tissue from the testis. This tissue, along with the contents of the cannula, are placed on a dry dish and inspected for the presence of sperm. The procedure can be repeated if the initial tissue is scanty or if no sperm are found. Finally, a tissue-cutting biopsy can also be performed to retrieve sperm percutaneously with the help of biopsy instruments (Tru-cut™ needle or Biopty™ gun) [31]. These needles are spring-activated, and are fired into the testis to retrieve a chip of tissue that can be analyzed for sperm. A short movie depicting the main steps of the procedure can be found at www.youtube.com/watch?v=o9MgknYEzN0.
6.3.3.2 Conventional TESE
The open or cTESE procedure can be performed through a single incision along the scrotal midline raphe. Alternatively, some surgeons may prefer doing two separate transverse hemiscrotal incisions. The dartos muscle and tunica vaginalis are then incised and the testis is delivered from the wound. A tunica albuginea “window” is made using a no. 10 surgical scalpel. The emerging testicular tissue is cut with microsurgery scissors. Hemostasis is then achieved using low-current electrocautery and the tunical opening is closed with nonabsorbable sutures. The tunica vaginalis, dartos muscle, and scrotal skin are then closed with absorbable sutures. The testicular tissue is placed on a Petri dish containing sperm-friendly media and is sent to the lab for processing.
6.3.3.3 Single Seminiferous Tubule Biopsy
This procedure is a modification of cTESE [7]. The testis is delivered through a scrotal wall incision as described above. Using a 26 G needle, multiple punctures can be made through avascular areas of the testicular surface. These punctures are then dilated with the tip of a microforceps, allowing a loop of seminiferous tubule to pop out. This loop is then pulled with the microforceps and examined under the microscrope. The number of puncture holes is determined by the quantity of retrieved sperm and the quality of the seminiferous tubules. Since the holes are very small in size, no suturing is needed.
6.3.3.4 Micro-TESE [32]
After delivering the testis from the scrotal incision, the tunica albuginea is examined under low magnification (6–8×) and a large horizontal midline incision is made through an avascular plane. The midline edges of the tunica albuginea are then clamped with small mosquito forceps to help in bivalving the testicle along its equatorial plane. The testicular parenchyma is then explored using high magnification (16–25×), looking for dilated seminiferous tubules (which presumably are lined by germ cells and are likely to contain sperm). The dissection is carried out in a systematic fashion and dilated seminiferous tubules are retrieved using microforceps from superficial and deep layers of the testicular parenchyma. If the testicular parenchyma appears homogenous, multiple random samples are retrieved from different areas of the testicular parenchyma. The samples are placed in a Petri dish containing sperm-friendly media and are sent to the laboratory for analysis. The tunica albuginea is then closed using nonabsorbable sutures in a running fashion. The remaining layers are closed with absorbable sutures. The wound is preferably injected with a local anesthetic (1 percent lidocaine) to reduce postoperative pain. A short movie depicting the main steps of the procedure can be found at www.brazjurol.com.br/videos/may_june_2013/Esteves_440_441video.htm [33].
6.3.4 Postoperative Care
All sperm retrieval procedures are performed in an outpatient setting and patients are generally discharged a few hours after the procedure. Patients are advised to apply scrotal ice packs and to stay in bed for the rest of the day of the procedure. After 24 hours, patients are advised to take a warm bath and wash the surgical site with soap and water. The incision should by dried with a sterile gauze and patients should be instructed to wear scrotal support for up to one week following surgery. Oral analgesics should be prescribed for 3–5 days after the procedure. Postoperative antibiotics are generally not required. Following TESA, patients can usually resume routine activities the following day; for open procedures, a sick leave of 3–7 days may be required. Strenuous physical activity and sexual intercourse should be avoided for up to 10 days following open procedures. Patients should be informed that swelling or ecchymosis of the surgical site can be expected to occur and will usually resolve within 1–2 weeks following surgery [34]. However, they should seek medical assistance if there is progressive pain, swelling, excessive bleeding, or discharge from the surgical site, or if they have a fever.
6.3.5 Complications of Testicular Sperm Retrieval
Persistent pain, infection, hydrocele, and hematoma are the most common complications that can occur following sperm retrieval procedures [4,5,35,36]. The complication rate varies according to the sperm retrieval technique performed. Since TESA is a blind technique, the risk of hematoma may be greater than for other procedures [36]. However, except for minor postoperative pain and swelling, no reports have indicated significant intra- or postoperative complications from TESA. Conventional TESE can have a detrimental effect on testicular function postoperatively, especially if large amounts of tissue were extracted [4,5,35]. Intratesticular hematomas were observed in up to 80 percent of patients undergoing scrotal ultrasound following testicular sperm extraction; however, this will often resolve spontaneously [36]. Micro-TESE is believed to offer a lower risk of complications as magnification will allow retrieval of less testicular tissue in addition to preservation of intratesticular blood supply. Nevertheless, transient reductions in serum testosterone are documented following micro-TESE and are believed to return to pre-surgical levels up to 12 months following the procedure [37].
6.4 Outcomes, Advantages, and Disadvantages of Different Surgical Sperm Retrieval Methods
6.4.1 Sperm Retrieval Outcomes
The testicular sperm retrieval rate for patients with NOA is influenced by a number of factors. While various predictors for testicular sperm retrieval have been recognized [38,39] and will be covered in detail in Chapter 10, the surgical procedure performed can significantly affect the sperm retrieval rate. The reported TESA retrieval rate ranges between 10 and 30 percent [36,40–44]. However, this rate increases to >65 percent in patients with previous positive TESA or with a testicular histopathology showing hypo-spermatogenesis [34,45]. The sperm retrieval rate with cTESE is about 50 percent [40], while that of micro-TESE ranges between 35 and 77 percent [34,46–53]. A systemic review by Deruyver et al. [54] compared the efficacy of micro-TESE to cTESE in men with NOA. The authors examined seven articles and reported a significantly higher sperm retrieval rate with micro-TESE (42.9–63 percent) in comparison to cTESE (16.7–45 percent). They also performed a sub-analysis of the sperm retrieval rate based on the histopathology result and observed a significantly better sperm retrieval rate with micro-TESE in patients with Sertoli-cell-only compared with cTESE. This result was echoed by another systematic review comparing three sperm retrieval methods: micro-TESE, TESA, and cTESE [40]. The results indicate that in patients with Sertoli-cell-only, micro-TESE is better than cTESE, while cTESE is better than TESA. Bernie et al. [55] conducted a meta-analysis of 15 studies to compare the outcomes of micro-TESE, cTESE, and TESA. In a direct comparison, the sperm retrieval rate was 1.5 times higher in micro-TESE compared with cTESE (95 percent CI 1.4–1.6), while it was two times higher in cTESE compared with TESA (95 percent CI 1.8–2.2). Due to the heterogeneity of the conducted studies, none of these reviews was able to compare the pregnancy outcomes between the different sperm retrieval methods.
6.4.2 Number of Samples
The number of biopsies that can be sampled during the different sperm retrieval techniques has been a subject of debate. Studies have indicated that up to four biopsies can be obtained from each testicle with TESA or cTESE [56,57]. One study reported that the mean number of biopsies necessary to retrieve sperm from patients with maturation arrest or Sertoli-cell-only was 4.2 ± 4.5 and 2.8 ± 2.5, respectively [58]. Since SST and micro-TESE do not produce major trauma to the testicular tissue, anywhere between 10 and 15 biopsies can be taken [57].
6.4.3 Choice of Surgical Sperm Retrieval Technique
The ideal surgical sperm retrieval method is the one that offers the highest sperm retrieval rate together with the lowest complication rate. The expense of the procedure is also an important factor to consider as some procedures are much more expensive than others. Therefore, the choice of surgical procedure should be tailored to each case. A number of clinical factors can aid in selecting the most appropriate procedure for a given case. Testicular histopathology, if previously known, is a good example as TESA may be offered for men with hypospermatogenesis, while micro-TESE may be a better option for those with Sertoli-cell-only. Testicular volume is another example as reports have indicated that micro-TESE is better than cTESE in patients with testicular volume <10 ml [59]. Many authors have advocated a staged surgical approach to sperm retrieval, in which they would start first with TESA and progress to SST or micro-TESE if no sperm were found with percutaneous aspiration [57,60].
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