Abstract
Sperm retrieval and intracytoplasmic sperm injection (ICSI) has become the natural treatment for couples with azoospermia-related infertility, and nowadays is also used for nonazoospermic indications. An increasing body of evidence overwhelmingly based on cohort studies has indicated that ICSI with ejaculated sperm of poor quality negatively affects the chances of assisted conception. Collectively, these data suggest that ICSI with testicular sperm is superior to ICSI with ejaculated sperm as a method of fertilization to overcome sperm DNA fragmentation-related infertility. The candidates are men with high sperm DNA damage in semen and those with severe oligozoospermia or cryptozoospermia. In these patients, percutaneous and open sperm retrieval are highly successful to harvest sperm, with few complications. Current evidence suggests the safe utilization of testicular sperm for ICSI in nonazoospermic men. Further research is warranted to confirm the clinical utility of this approach as a routine ART treatment.
8.1 Introduction
The development of intracytoplasmic sperm injection (ICSI) was an extraordinary achievement in the field of assisted reproduction technology (ART) [1]. The method was initially introduced in 1992 as a modification of conventional in vitro fertilization (IVF), enabling men with low sperm quantity and quality to father a biological child [2]. Nowadays, ICSI has become the most commonly used method of fertilization in ART, and it is the method of choice for overcoming untreatable severe male factor infertility [3].
8.1.1 Sperm Retrieval in Azoospermic Men
A few years after the introduction of ICSI, methods to harvest sperm from the epididymides and testes were developed to help men with azoospermia overcome infertility (reviewed in [4]). Azoospermia is a condition that affects approximately 10–15 percent of men with infertility and is characterized by a complete absence of spermatozoa in the ejaculate [5]. Before ICSI, few options were available for the affected men to father a biological child, in particular, for those with nonobstructive azoospermia [6]. The latter is associated with untreatable testicular disorders that result in spermatogenic failure. Nevertheless, 30–60 percent of men with nonobstructive azoospermia have focal testicular sperm production that can be retrieved and used for ICSI [7]. By contrast, obstructive azoospermia results from bilateral obstruction of the seminal ducts. Spermatogenesis is intact in men with obstructive azoospermia; therefore, sperm can be retrieved from epididymides or testicles in virtually all cases [8].
Nowadays, the two methods most commonly used to harvest sperm in men with azoospermia are percutaneous acquisition and open surgery (with or without the aid of microsurgery) [9]. In men with obstructive azoospermia, the sperm retrieval technique and the cause of obstructive azoospermia have little impact on sperm retrieval success and ICSI outcome [8]. Among men with nonobstructive azoospermia, the use of microsurgical TESE (testicular sperm extraction) yields higher sperm retrieval success rates and fewer complications than conventional TESE (reviewed in [7]). After retrieval of epididymal or testicular sperm, ICSI is used instead of conventional IVF as the retrieved gametes are unable to fertilize the oocytes by conventional IVF.
8.1.2 Sperm Retrieval in Non-azoospermic Men
Ejaculated sperm are generally regarded as having the highest fertilization potential as they have completed their transit through the male reproductive tract. Furthermore, the early ICSI experience suggested that sperm parameters – evaluated on ejaculated semen; namely, concentration, motility, and morphology – had no significant influence on outcomes [10]. However, as experience accumulated, reports of an association between sperm quality and ICSI outcomes increased steadily [11–13]. Concerns of a possible role of the paternal gamete on poor ICSI outcomes have been raised, in particular, in cases where (1) ejaculated specimens containing abnormal levels of sperm with damaged chromatin were used for sperm injections; and (2) the number (and quality) of ejaculated sperm was too low (e.g., cryptozoospermia, which is denoted by absence or very few spermatozoa in the fresh ejaculate but observed after microscopic examination of centrifuged pellet).
8.1.2.1 Biological Plausibility
It is well-established that sperm chromatin integrity is vital for the birth of healthy infants [14]. In ART, fertilization of oocytes by sperm with damaged chromatin might lead to an increased risk of fertilization failure, embryo development arrest, implantation failure, miscarriage, congenital malformations, as well as perinatal and postnatal morbidity [15–17]. The causes of sperm chromatin damage include apoptosis during spermatogenesis, deficient chromatin remodeling during spermiogenesis [18], activation of endogenous caspases and endonucleases [19], exogenous factors such as environmental toxicants, radiotherapy, and chemotherapy [20], and oxidative stress [21]. In normal conditions, there is an equilibrium between reactive oxygen species (ROS) production and antioxidant defense system in the male reproductive tract. When ROS production overwhelms antioxidant defenses, a state of oxidative stress ensues. As a result, excessive ROS attack both sperm membranes and nuclear and mitochondrial DNA, mostly during sperm transit through the male reproductive tract [20–22].
Non-azoospermic infertile men often have high levels of sperm chromatin damage in their neat semen [23,24]. Abnormal levels of sperm chromatin damage have been demonstrated primarily in ejaculates of men with poor conventional semen parameters (count, motility, morphology), although counterparts with semen analysis within normal ranges might also be affected [25,26]. Varicocele, systemic diseases, male accessory gland infections, advanced paternal age, obesity, lifestyle and environmental factors, radiation, and heat exposure are some of the conditions associated with sperm chromatin damage (reviewed in [27]). Most of these stressors share oxidative stress as a common trait.
However, data from human studies indicate that sperm chromatin damage is lower in testicular sperm than in epididymal and ejaculated sperm. This evidence derives from studies involving men with obstructive azoospermia as well as non-azoospermic counterparts, as discussed below.
In a study involving men with obstructive azoospermia, Steele et al. observed that the frequency of sperm with intact chromatin – assessed by the Comet assay – in paired samples of testicular and epididymal spermatozoa was higher in the former (83.0 ± 1.2 percent versus 75.4 ± 2.3 percent, p < 0.05). [28]. In a similar study involving 25 men with obstructive azoospermia, O’Connell et al. showed the testicular sperm had better quality than epididymal sperm [29]. The authors used polymerase chain reaction (PCR) and alkaline Comet assay to assess mitochondrial DNA (mtDNA) and nuclear DNA (nDNA), respectively. Testicular sperm had significantly more wild-type mtDNA and lower incidence of multiple deletions and smaller mtDNA fragments than epididymal sperm, whereas epididymal sperm displayed more large-scale deletions (p < 0.05). In their study, a strong correlation was found between nuclear DNA damage, the number of mtDNA deletions (r = 0.48, r = 0.50, p < 0.0001) and deletion size (r = 0.58, r = 0.60, p < 0.001) in both epididymal and testicular sperm. Lastly, Hammoud et al., using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay, assessed the levels of sperm chromatin damage in paired testicular and epididymal specimens of 21 men with obstructive azoospermia [30]. Sperm chromatin damage values were lower in the testis (6.7 ± 0.7 percent) than in the epididymis (caput: 14.9 ± 1.9 percent; p = 0.0007; and corpus/cauda: 32.6 ± 3.1 percent; p < 0.0001).
Likewise, studies assessing paired testicular and ejaculated specimens of non-azoospermic men demonstrate better chromatin integrity in the former. In 2005, Greco et al. published the first series showing that the rates of sperm exhibiting chromatin damage was lower in testicular sperm than ejaculated sperm (TUNEL assay; testis: 4.8 ± 3.6 percent [testis] versus 23.6 ± 5.1 percent [ejaculate]) [31]. Later, in 2010, Moskovtsev et al. confirmed these findings in a group of men with persistent high sperm chromatin damage in neat semen after use of oral antioxidants (TUNEL assay: 13.3 ± 7.3 percent [testis] versus 39.7 ± 14.8 percent [ejaculate], p < 0.001) [32]. Another cohort study by the same authors published in 2012 showed similar results (TUNEL assay: 14.9 percent [testis] versus 40.6 percent [ejaculate]) [33]. In 2015, Esteves et al. used the sperm chromatin dispersion (SCD) assay to evaluate paired testicular and ejaculated specimens of a large cohort of men with idiopathic oligozoospermia and high rates of sperm chromatin damage on neat ejaculate [34]. In their study, testicular sperm had fivefold lower rates of sperm chromatin damage than ejaculated sperm (8.3 ± 5.3 percent versus 40.7 ± 9.9 percent, respectively; p < 0.001). In another 2015 cohort study of men with severe oligozoospermia, Mehta et al. also showed less chromatin damage in testicular sperm compared to ejaculated sperm (TUNEL assay: 5 percent versus 24 percent, respectively; p < 0.001) [35]. A 2017 systematic review – followed by meta-analysis – compiled the results of these five studies [36] and showed that the mean difference (MD) in sperm DNA fragmentation rates – a measure of sperm chromatin damage – was −24.6 percent (95 percent CI –32.5 to –16.6; I2 = 92 percent; p < 0.001). Four studies used the TUNEL assay for the assessment of SDF (pooled MD: −19.8 percent; 95 percent CI −22.3 to −17.2; I2 = 15 percent; p < 0.001), whereas one study used the SCD assay (MD: −32.4 percent; 95 percent CI −34.85 to −29.95; p < 0.001).
In conclusion, fair evidence indicates that among infertile men, sperm chromatin integrity progressively decreases as sperm transit across the genital tract. The mechanisms are not fully understood, but apparently, oxidative attack after sperm release from the seminiferous tubules is the main reason for the higher frequency of sperm with damaged chromatin in both epididymal and ejaculated sperm than testicular sperm. These findings, combined with the observations of possible improved ICSI outcomes with the use of sperm retrieved from the testes in preference over ejaculated sperm – discussed in the next sections – have led to the extended indications of sperm retrieval.
8.2 Indications
Table 8.1 lists the indications for sperm retrieval in non-azoospermic infertile males.
| Type of male infertility | Fertilization method | Level of evidence | References | Grade of recommendation |
|---|---|---|---|---|
| Elevated levels of sperm with chromatin damage on neat ejaculate§ | ICSI mandatory | 2a 2b 4 | 36 34, 37–40 19, 30, 34, 41 | B–C |
| Severe oligozoospermia and cryptozoospermia* | ICSI mandatory | 2b 3a 3b 4 | 42–44 45, 46 47–49 50 | B–C |
ICSI, intracytoplasmic sperm injection.
§ Assessed by the four most common assays, namely TUNEL, SCD, Comet, and sperm chromatin structure assay (SCSA); in this scenario, testicular sperm retrieval could be offered as a means to improve ICSI outcomes, in particular for men with persistent high levels of sperm with damaged chromatin on the neat ejaculate – despite all efforts to treat any cause associated with sperm chromatin damage.
* No or extremely rare spermatozoa in the fresh ejaculate but observed after microscopic examination of centrifuged pellet.
Level 2a: systematic review with homogeneity of cohort studies; homogeneity defined by lack of worrisome variations (heterogeneity) in the directions and degrees of results between individual studies.
Level 2b: Individual cohort study (including low-quality randomized controlled trials [RCT]).
Level 3a: Systematic review with homogeneity of case–control studies; homogeneity defined by lack of worrisome variations (heterogeneity) in the directions and degrees of results between individual studies.
Level 3b: Individual case–control study.
Level 4: Case series and poor-quality cohort and case–control studies. Poor-quality cohort studies denoted by those that failed to clearly define comparison groups and/or failed to measure exposures and outcomes in the same (preferably blinded), objective way in both exposed and non-exposed individuals and/or failed to identify or appropriately control known confounders and/or failed to carry out a sufficiently long and complete follow-up of patients. Poor-quality case–control studies denoted by those that failed to clearly define comparison groups and/or failed to measure exposures and outcomes in the same (preferably blinded), objective way in both cases and controls and/or failed to identify or appropriately control known confounders.
Grade B: consistent level 2 or 3 studies or extrapolations from level 1 studies (systematic reviews with homogeneity of RCT and individual RCT with narrow confidence interval).
Grade C: level 4 studies or extrapolations from level 2 or 3 studies. Extrapolations are where data is used in a situation that has potentially clinically important differences to the original study situation.
Grade D: level 5 evidence (denoted by expert opinion without explicit critical appraisal, or studies based on physiology, bench research or “first principles”) or troublingly inconsistent or inconclusive studies of any level.
Levels of evidence and grades of recommendation according to ‘Oxford Centre for Evidence-based Medicine – Levels of Evidence (March 2009)’, available at: www.cebm.net/2009/06/oxford-centre-evidence-based-medicine-levels-evidence-march-2009.
8.3 Protocol
Both percutaneous and open sperm retrieval procedures can be used to harvest sperm from the seminiferous tubules in non-azoospermic men. These methods are commonly carried out on an outpatient basis and the same day as oocyte retrieval.
8.3.1 Percutaneous
8.3.1.1 Testicular Sperm Aspiration
Testicular sperm aspiration (TESA) is carried out under local anesthesia applied to the spermatic cord, intravenous sedation or general anesthesia (Figure 8.1) [4]. The testicle is held firmly and punctured using a large gauge needle (e.g., 18 G) attached to a syringe. The needle is introduced at an oblique angle in its anterior aspect of the upper pole to decrease the risk of vascular injury. Loupe magnification may be used during puncture to avoid transfixing scrotal vessels seen through the skin [6]. Negative pressure is applied using different maneuvers (e.g., Cameco syringe holder; Figure 8.1) to aid extraction of seminiferous tubules. The seminiferous tubules are disrupted by moving the needle back and forth so they can be easily aspirated. The sample is immediately analyzed in the IVF laboratory and, if adequate, the procedure is finished; otherwise, the contralateral testis is punctured at the same operative time. A short movie depicting the main steps of the procedure can be found at www.youtube.com/watch?v=o9MgknYEzN0.
Figure 8.1 Testicular sperm aspiration. A 18 G needle connected to a 20 ml syringe fit to the Cameco holder is percutaneously inserted into the testis. Negative pressure is created, and the tip of the needle is moved within the testis to disrupt the seminiferous tubules and sample different areas.
8.3.2 Open Non-microsurgical
8.3.2.1 Testicular Sperm Extraction
Testicular sperm extraction is carried under local anesthesia, intravenous sedation combined with local anesthesia, spinal block, or general anesthesia [6]. The procedure can be performed with or without testis delivery. The skin and subjacent layers are incised transversally to expose the tunica albuginea, which is opened with a sharp knife. Typically, a small transversal albuginea opening (0.5–1.0 cm incision) is made at the mid-testicular pole, and one or multiple fragments of the parenchyma is cut off with the aid of scissors or forceps [51] (Figure 8.2). The tunica albuginea is closed with either non-absorbable or absorbable sutures, whereas the tunica vaginalis, dartos, and skin are sutured with absorbable suture.
Figure 8.2 Testicular sperm extraction (TESE). Single or multiple incisions are made on the tunica albuginea and one or several testicular biopsies are taken. Extracted specimens are placed on a Petri dish with culture media and sent to the laboratory for processing by mechanical mincing under the stereomicroscope. The cell suspension is examined under the inverted microscope for sperm search. After confirmation of an adequate number of sperm for ICSI, specimens are incubated at room temperature until sperm injections take place.
8.3.3 Open Microsurgical
8.3.3.1 Microdissection Testicular Sperm Extraction
Microdissection testicular sperm extraction (micro-TESE) – initially described by Schlegel in 1996 – can be performed under intravenous sedation combined with local anesthesia, general anesthesia or spinal block [6,51,52]. After skin incision, the testis is delivered outside the scrotum. The tunica albuginea is widely incised transversally, and the testicular parenchyma is exposed (Figure 8.3). The operating microscope with optical magnification ranging of 16–25× is used, and dissection of seminiferous tubules is carried out in search of enlarged tubules that are more likely to contain mature sperm. During dissection, damage to the intratesticular blood supply is actively avoided. Extracted specimens are transferred to the IVF laboratory for examination. 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 [53].
Figure 8.3 Microsurgical testicular sperm extraction. After the testicle is exteriorized, a single and large incision is made in an avascular area of the albuginea to expose the seminiferous tubules. The dilated tubules are identified and removed with micro-forceps. The illustration in the middle of the figure depicts histopathology cross-sections of a dilated seminiferous tubule with active spermatogenesis and a thin tubule with germ cell aplasia.
8.3.4 Laboratory Sperm Handling
Methods to remove cellular debris and red blood cells are generally used to prepare the extracted specimens for ICSI. Since iatrogenic sperm DNA damage can occur during sperm processing, all efforts should be made to minimize such damage. Controlling centrifugation force and duration, limiting exposure to ultraviolet light and temperature variation, optimizing laboratory air quality conditions, and using high-quality reagents, culture media, and disposable materials are critical elements [54,55]. Whenever possible, techniques aimed at improving the sperm fertilizing potential should be applied, including the use of chemical stimulants and/or methods to select viable sperm for ICSI. The latter is particularly important when only immotile spermatozoa are harvested. Table 8.2 provides an overview of the laboratory processes concerning the processing of surgically extracted specimens. A detailed laboratory procedure for processing such specimens can be found elsewhere [56].
Table 8.2 Laboratory strategies to handle testicular specimens retrieved from non-azoospermic men
| Process | Procedures | Techniques | Main goal | Critical level |
|---|---|---|---|---|
| Testicular tissue processing | Mechanical tissue mincing | Disruption of seminiferous tubules using fine needles or micro-scissors, and forced pass through small diameter catheters | Tubular breakdown and cellular content loss | Critical |
| Red blood cell lysis | Incubation of testicular suspensions with erythrocyte lysing buffer solution | Removal of excessive blood cells from testicular specimens | Optional | |
| Motility enhancement | Incubation of testicular specimens with pentoxifylline | Selection of viable sperm for ICSI | Optional | |
| Laboratory environment and laboratory practices | Air quality control | Air particulate and volatile organic compounds filtration | Secure optimal safety conditions for gamete handling, sperm injection, and embryo culture | Recommended |
| Maintenance of temperature and pH stability | Quality control and quality assurance of instruments, equipment, and reagents | Avoid iatrogenic cellular damage | Critical | |
| Centrifugation | Simple washing with buffered medium or mini-gradient centrifugation using low centrifugation forces | Avoid iatrogenic cellular damage | Critical | |
| Sterile techniques | Manipulation of gametes and embryos in laminar flow cabinets or inside controlled environments | Secure optimal safety conditions for gamete handling, sperm injection, and embryo culture | Critical | |
| Intracytoplasmic sperm injection | Sperm selection | Hypo-osmotic swelling test, mechanical touch technique, and laser-assisted sperm selection | Selection of viable immotile sperm for ICSI | Optional |
Importantly, ICSI should be carried out immediately after completion of sperm processing. Prolonged sperm incubation, in particular, at 37 °C, and sperm freezing might negatively affect sperm chromatin damage [22,57,58]. Therefore, sperm retrieval for non-azoospermic men should be scheduled on the same day as oocyte retrieval.
8.4 Outcomes
8.4.1 Sperm Retrieval Success Rates
Sperm retrieval success rates in the context of non-azoospermic men are virtually 100 percent [36]. Such men are either oligozoospermic or normozoospermic, thus exhibiting relatively well-preserved sperm production. The seminiferous tubules of men with normozoospermia invariably contain mature sperm; therefore, both percutaneous and open testicular sperm retrieval can be used with similar success rates [59]. By contrast, men with oligozoospermia show varying degrees of sperm production and the choice of the sperm retrieval technique might play a role, particularly for men with severe oligozoospermia. While similar success rates can be achieved by percutaneous and open sperm retrieval methods in men with mild and moderate oligozoospermia [34], open methods, in particular microsurgical ones, should be preferred in men with severe oligozoospermia to increase the likelihood of harvesting sperm and decrease the risk of complications [35].
8.4.2 Sperm Retrieval Complications
Sperm retrieval complications include pain, swelling, infection, hematoma, and loss of testicular function [4,51]. The complication rates are reported to be less than 5 percent overall, most of which resolve spontaneously without compromising testicular function [51,60]. A transient decline in testosterone levels is expected after open procedures, which tend to return to preoperative values in approximately 6–12 months [60]. However, large-volume tissue extraction, mainly using non-microsurgical multiple biopsy TESE, can cause testicular damage (reviewed in [7]). Testicular atrophy and permanent reduction in androgen production have been occasionally reported after TESE, which might cause hypogonadism [61].
Nevertheless, most of the data mentioned above relate to studies involving men with azoospermia. In the context of non-azoospermic men, sperm retrieval is associated with very few complications as minimal tissue extraction yields sufficient numbers of sperm for ICSI [62]. Yet, given the potential risk for complications and adverse effects on testicular function, sperm retrieval should be performed by well-trained urologists.
8.4.3 Pregnancy Outcomes
8.4.3.1 Sperm chromatin damage
In 2005, Greco et al. published the first successful series of ICSI using testicular sperm in 18 non-azoospermic patients with high sperm chromatin damage on neat semen [31]. On the day of oocyte retrieval, the male partners underwent TESA or TESE. In this series, intracytoplasmic testicular sperm injection resulted in eight clinical pregnancies (44.5 percent), whereas only one pregnancy (5.6 percent) that ended in miscarriage had been obtained in previous cycles.
Later in 2010, Sakkas and Alvarez reported on 72 patients with high levels of sperm chromatin damage on neat semen who used testicular sperm for ICSI [20]. Testicular sperm aspiration was used to harvest sperm from the seminiferous tubules. In this series, the clinical pregnancy rates and implantation rates were higher after ICSI using testicular sperm than ejaculated sperm (40.0 percent and 28.1 percent versus 13.8 percent and 6.5 percent, respectively; p < 0.05).
In 2015, Esteves et al. reported the first prospective comparative study investigating the efficacy of use of testicular sperm for ICSI among men with idiopathic oligozoospermia and high sperm chromatin damage in neat semen [34]. On the day of oocyte retrieval, either TESA or conventional TESE were used for sperm retrieval. In this series that included 172 patients with no history of ICSI failure, the live birth rate was higher with testicular sperm (versus ejaculated sperm), with an adjusted relative risk of 1.76 (95 percent CI 1.15–2.70; p = 0.008). The authors reported that approximately five patients had to be treated to result in one additional live birth by testicular sperm for ICSI compared to ejaculated sperm.
Also in 2015, Mehta et al. published another successful series of ICSI using sperm retrieved from the testis of 24 patients with severe oligozoospermia and history of failed ICSI [35]. The male partners underwent micro-TESE, in which the seminiferous tubules were dissected, mature sperm were identified, and the extracted sperm were used for ICSI. In this series, 12 clinical pregnancies were achieved, all of which resulted in live births.
In a 2016 large retrospective cohort study evaluating ICSI outcomes in patients with high sperm chromatin damage on neat semen in Australia, Bradley et al. reported that ICSI with testicular sperm resulted in higher live birth rates (49.8 percent) than ICSI with ejaculated sperm, even when advanced laboratory methods – intracytoplasmic morphologically selected sperm injection (IMSI) and hyaluronic acid sperm selection ICSI (PICSI) – were used to select ejaculated sperm with better chromatin integrity for ICSI (38.3 percent and 28.7 percent, respectively; p = 0.02) [37].
Later in 2017, Pabuccu et al. investigated the efficacy of use of testicular sperm for ICSI among normozoospermic men and high sperm chromatin damage in neat semen [38]. Seventy-one couples with a history of ICSI failure were included. On the day of oocyte retrieval, TESA was used to harvest sperm from the seminiferous tubules. In this series, ICSI with testicular sperm was associated with higher rates of clinical pregnancy (41.9 versus 20.0 percent; p = 0.04) and ongoing pregnancy (38.7 versus 15.0 percent; p = 0.02) than with ejaculated sperm.
A systematic review published in 2017 aggregated the evidence of the studies mentioned above concerning the use of testicular sperm for ICSI in men with high sperm chromatin damage in semen. A total of 507 ICSI cycles in which 3840 oocytes were injected with either ejaculated sperm or testicular sperm was included. Using meta-analysis, the authors showed higher clinical pregnancy rate (OR 3.6, 95 percent CI 1.94–6.69; I2 = 0 percent; p < 0.0001) and live birth rate (OR 2.6, 95 percent CI 1.54–4.35, I2 = 0 percent; p = 0.0003), and lower miscarriage rates (OR 0.40, 95 percent CI 0.10–1.65, I2 = 34 percent; p = 0.005) when comparing Testi-ICSI with ejaculated ICSI [36].
Subsequently, in 2018, Arafa et al. assessed ICSI outcomes with the use of testicular sperm in 36 men with high sperm chromatin damage in semen [41]. The male partners had either normozoospermia or oligozoospermia, and TESA was used for sperm retrieval. The couples reported a history of ICSI failure with use of ejaculated sperm. In this series, the rates of clinical pregnancy were higher with Testi-ICSI than ejaculated sperm ICSI (38.9 versus 13.8 percent; p < 0.0001). Of these pregnancies, 17 were reported to result in live offspring in the testicular sperm ICSI group compared to only three in the ejaculated sperm ICSI group.
Also in 2018, Zhang et al. compared ICSI outcomes by testicular sperm versus ejaculated sperm in a group of 102 infertile men [39]. Like the reports mentioned above, the male partners had high rates of sperm chromatin damage in neat semen. The rates of clinical pregnancy (36.0 versus 14.6 percent; p = 0.017) and delivery (36.0 versus 9.8 percent; p = 0.001) were higher after the transfer of embryos resulting from Testi-ICSI than ejaculated sperm for ICSI.
Lastly, in 2019, Herrero et al. investigated the use of Testi-ICSI in 145 couples with no previous live births and a history of at least two previous failed ICSI cycles with ejaculated sperm [40]. The studied men had high levels of sperm chromatin damage on neat semen, and TESE was the method used to harvest sperm from the seminiferous tubules. In this series, cumulative live birth rates were higher after use of Testi-ICSI than ejaculated sperm for ICSI (21.7 versus 9.1 percent; p < 0.01).
Collectively, data from six retrospective studies and three prospective studies, including a total of 830 patients and 902 ICSI cycles, suggest that ICSI with testicular sperm is superior to ICSI with ejaculated sperm to overcome infertility among non-azoospermic men with high sperm chromatin damage in semen (Table 8.3). Testi-ICSI has been postulated to bypass post-testicular sperm chromatin damage caused by oxidative stress during sperm transit through the epididymis. As a result, the chances of oocyte fertilization by genomically intact testicular spermatozoa are increased, thus resulting in an increased possibility of formation of a normal embryonic genome and an increased likelihood of achieving a live birth. However, no RCT has yet investigated the efficiency of testicular sperm in non-azoospermic men with sperm chromatin damage.
Table 8.3 Characteristics and main outcome measures of studies reporting ICSI outcomes with testicular versus ejaculated sperm in non-azoospermic men with high sperm DNA fragmentation in the semen
| Study characteristics | Indication | Sperm retrieval method | Outcomes | ||||||
|---|---|---|---|---|---|---|---|---|---|
| Reference | Design | Subjects and cohort size (N) | Test used for sperm chromatin damage assessment and cut-off values (%) | Paired SDF results in testicular and ejaculated sperm (%) | Sperm retrieval method | Sperm retrieval success and complication rates (%) | Fertilization rate (%) | Clinical pregnancy rate (%) | Ongoing pregnancy or live birth rates5 (%) |
| 31 | Case series | Predominantly normozoospermic infertile men (18) Couples with history of ICSI failure performed with ejaculated sperm | TUNEL (15) | 23.6 ± 5.1 (E) and 4.8 ± 3.6 (T) (p < 0.001) | TESE and TESA | 100 and NR | 74.91 | 44.42 | NR |
| 20 | Case series | Couples with history of IVF/ICSI failure (68) with ejaculated sperm | TUNEL (20) | NR | TESA | NR | 58.0; range: 20–100 | 40.0 | NR |
| 34 | Prospective cohort | Oligozoospermic (sperm concentration 5–15 million/ml) infertile men (172) Couples with no history of ICSI failure (Testi-ICSI, n = 81 and Ejac-ICSI, n = 91) | SCD (30) | 40.9 ± 10.2 (E) and 8.3 ± 5.3 (T) (p < 0.001) | TESE and TESA | 100 and 6.2 | 69.4 (E) vs. 56.1 (T) (p = 0.0001) | 40.2 (E) vs. 51.9 (T) (NS) | LBR: 26.4 (E) vs. 46.7 (T) (p = 0.007) |
| 35 | Case series | Oligozoospermic (sperm concentration <5 million/ml) infertile men (24) Couples with one or more failed IVF or ICSI cycles using ejaculated sperm | TUNEL (7) | 24.0 (9% CI: 19–34) (E) and 5.0 (95% CI: 3–7) (T) (p = 0.001) | Micro-TESE | 100 and NR | 54.0 | 50 | 50 |
| 49 | Retrospective cohort | Predominantly oligozoospermic men infertile men*; Testi-ICSI (n = 148), Ejac-ICSI (n = 80) | SCIT (29) | NR | TESE and TESA | NR | 66.0 (E) vs. 57.0 (T) (p < 0.001) | 27.5 (E) vs. 49.5 (T) (p < 0.01) | 24.2 (E) vs. 49.8 (T) (p < 0.05) |
| 50 | Retrospective cohort | Normozoospermic infertile men (71) Couples with history of ICSI failure using ejaculated sperm (Testi-ICSI, n = 31; Ejac-ICSI, n = 40) | TUNEL (30) | 41.7 ± 8.2 (E) | TESA | 100 and NR | 74.1 ± 20.7 (T) and 71.1 ± 26.9 (E) (NS) | 41.9 (T) and 20.0 (E) (p = 0.04) | OPR: 38.7 (T) vs. 15.0 (E) (p = 0.02) |
| 51 | Prospective cohort; interventions applied in the same patients | Oligozoospermic and normozoospermic infertile men (36) Couples with history of ICSI failure performed with ejaculated sperm | SCD (30) | 56.3 ± 15.3 (E) | TESA | 100 and NR | 46.4 (T) and 47.8 (E) (NS) | 38.9 (T) and 13.8 (E) (p < 0.0001) | LBR: 38.9 (T) vs. 8.0 (E) (p < 0.0001) |
| 52 | Prospective cohort4 | Oligozoospermic and normozoospermic infertile men (102) Couples with no history of ICSI failure (Testi-ICSI, n = 61; Ejac-ICSI, n = 41) | SCSA (30) | NR | TESA | 100 and NR | 70.4 (T) vs. 75.0 (E) (NS) | 36.0 (T) vs. 14.6 (E) (p = 0.01) | LBR: 36.0 (T) vs. 9.8 (E) (p = 0.001) |
| 53 | Retrospective cohort | Couples with no previous live births and a history of at least two previous failed ICSI cycles with ejaculated sperm (Testi-ICSI, n = 77; Ejac-ICSI, n = 68) | SCSA (≥25); TUNEL (≥36) | NR | TESE | NR | DFI ≥25 (SCSA): 66.3 (T); 62.9 (E) (NS) DFI ≥36 (TUNEL): 61.2 (T); 57.6 (E) (NS) | DFI ≥25 (SCSA): 18.2 (T); 9.1 (E) (p < 0.02) DFI ≥36 (TUNEL): 23.1 (T); 0.0 (E) (p < 0.02) | 3DFI ≥25 (SCSA): 21.7 (T); 9.1 (E) (p < 0.01) DFI ≥36 (TUNEL): 20.0 (T); 0 (E) (p < 0.02) |
* Number of ICSI cycles; E, ejaculated sperm group; Ejac-ICSI, ICSI with ejaculated sperm; LBR, live birth rate; micro-TESE, microdissection testicular sperm extraction; NR, not reported; NS, not significantly different; OPR, ongoing pregnancy rate; SCD, sperm chromatin dispersion test; SCIT, sperm chromatin integrity test, a variation of sperm chromatin structure assay (SCSA); SDF, sperm DNA fragmentation; T, testicular sperm group; Testi-ICSI, ICSI with testicular sperm; TESE, Testicular sperm extraction, TESA, testicular sperm aspiration; TUNEL, terminal deoxyribonucleotide transferase-mediated dUTP nick-end labeling assay
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