Testicular mapping-guided sperm retrieval vs. upfront microTESE in non-obstructive azoospermia: a comparison of sperm retrieval, pregnancy and live-birth rates

Introduction

Background

Non-obstructive azoospermia (NOA) poses a multifaceted challenge to reproductive urologists. Men with NOA can often conceive a biological child, but the treatment pathway carries a high level of uncertainty, which adds complexity to patient counseling. Unfortunately, a non-invasive predictor of successful sperm retrieval (SR) with high sensitivity and specificity has not been identified (1-3). Measures such as follicle stimulating hormone (FSH), inhibin B, and testicular volume may suggest spermatogenic failure, but do not rule out the possibility of procuring sperm via testicular microdissection Microscopic Testicular Sperm Extraction (microTESE) (4,5). Testicular histology from diagnostic biopsy is recognized as a predictor of successful SR; however, the limitations of diagnostic biopsy include high discordance in intra-observer pathologic diagnosis and inadequate sensitivity arising from the focality of spermatogenesis in NOA (6,7).

Sperm extraction by microTESE has thus become the standard of care for SR in men with NOA. Since its introduction in 1999, microTESE has demonstrated the highest success compared to conventional percutaneous and open SR techniques (8-10). Despite refinements since its introduction, microTESE remains an invasive and costly procedure that carries a 40–50% chance of SR failure (11). A less invasive predictor of successful SR would be helpful for counseling men with NOA, in order to avoid the morbidity of microTESE (1).

Fine needle aspiration mapping (FNAM) of the testis was first described in 1965 and is a robust predictor of SR success in NOA (12-15). FNAM is a purely diagnostic procedure that employs cytologic analysis to systematically evaluate the presence of mature spermatozoa at many sites throughout the testes and yields high concordance with testicular biopsy histology. However, FNAM improves sensitivity for the detection of mature spermatozoa and may reveal focal sperm production in areas remote from a traditional biopsy in up to 1/3 of NOA cases (15-21). As such, FNAM improves counseling of men with NOA regarding SR prognosis with either microTESE or less invasive retrieval techniques.

Rationale and knowledge gap

Whether upfront microTESE or FNAM-guided SR is employed in the urologic community has largely been dictated by institutional or surgeon preferences. No direct comparison of these two pathways in a single cohort of patients with NOA exists. Key questions regarding the outcomes of these two pathways remain unanswered due to practical limitations inherent to the conduct of randomized controlled trials in male infertility.

Owing to our collaborative institutional design, wherein multiple reproductive urologists and reproductive endocrinologists practice together in concert with a single high-volume andrology laboratory, the two management pathways of NOA have been utilized concurrently based upon surgeon training and preference.

Objective

The primary objective of this study was to perform a direct comparison of SR, pregnancy, and live-birth rates following FNAM-guided SR and upfront microTESE performed by three reproductive urologists at a single fertility clinic. Our secondary objectives were to describe the FNAM positivity rate and histologic patterns, the influence of FNAM on SR technique, and SR success rates by type of SR technique. We present this article in accordance with the STROBE reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-24-362/rc).

Methods

A retrospective review of patient records from 2010 to 2019 was conducted. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was conducted with approval from the University of Washington Institutional Review Board (No. 00004466) and informed consent was waived due to its retrospective nature.

Patients

All men diagnosed with azoospermia according to the International Classification of Disease (ICD-10) were cross-referenced with those who underwent FNAM, testis biopsy or testicular SR (microTESE, TESE, TESA) based upon procedural codes as initial surgical management. Confirmation of NOA was made by review of semen analyses (including post-centrifugation analysis), chart notes and laboratory data. Patients who did not meet the diagnostic criteria for NOA (e.g., obstructive azoospermia or congenital bilateral atresia of the vas deferens) or who were positive for Y chromosomal microdeletions (YCMD) in AZFa or AZFb were excluded. Patients with Klinefelter’s syndrome were also excluded as they universally underwent microTESE due to small testicular volume precluding FNAM. AZFc microdeletion was included in the final analysis. FNAM results were verified by andrology reports, and surgical SR and microTESE operative reports were reviewed to determine success. Chart review of linked partner was performed in order to corroborate SR success, and to review results of in-vitro fertilization and intracytoplasmic sperm injection (IVF/ICSI) procedures, pregnancy and live-birth data.

FNAM technique and interpretation

FNAM was performed as previously described under local anesthesia (15). After sterile preparation, the spermatic cord and overlying scrotal skin is infiltrated with local anesthesia. The testis is then elevated, and a moist gauze tourniquet wrapped taut to secure the testis in its anatomic orientation with the epididymis posteriorly. The aspiration sites are marked with four rows of three, each approximately 2–3 mm apart, adjusted according to testis size. A total of 12 FNAM sites are planned on each testis. FNA is performed with a beveled 1-inch, 23-gauge needle with the use of a suctioning syringe holder (Cameco Syringe Pistol, Becton-Dickinson Co., Franklin Lakes, NJ, USA). Precise, suction-cutting, oscillating movements (10–20×) allow for gentle tissue aspiration. Aspirated tissue is dispensed onto a slide, smeared, allowed to air dry, then fixed in methanol. Slides are stained with Hemacolor stain set.

Adequacy of tissue sampling is determined based upon criteria set forth by Turek et al., whereby at least 12 clusters of testicular cells or at least 2,000 well-dispersed testicular cells are required (15). Individual slides are scrutinized for the presence or absence of mature spermatozoa by an experienced andrologist. Slides are also scrutinized for germ cells arrested at the secondary spermatocyte or spermatid in the absence of mature spermatozoa, in which case a diagnosis of maturation arrest (MA) is assigned. Finally, in the absence of any form of germ cells, a diagnosis of Sertoli cell only (SCO) or germ cell aplasia is made. In the application of positive FNAM findings to clinical use, the semi-quantitative diagnoses of diffuse and focal hypospermatogenesis are employed. If 6 or more positive sites per testis and at least 3 positive contiguous sites are identified with reduced spermatozoa, diffuse hypospermatogenesis is presumed. Focal hypospermatogenesis is characterized by significant heterogeneity or fewer than 6 positive sites per testis with reduced spermatozoa.

Upfront microTESE technique

All microTESEs were performed under general anesthesia, as previously described (8,22). In the case of upfront microTESE, the larger of the two testes is explored first. After delivering the testis, an equatorial incision of the tunica albuginea is made to “bivalve” the testis. The testis is systematically evaluated under high-powered microscopy, searching for dilated tubules. The same andrology team performs real time examination of the harvested, morcellated tissue in real time. If sperm are identified, adequacy of the sample is corroborated by the embryologist. If sperm are not found after systematic exploration of one testis, the procedure is repeated on the contralateral testes.

FNAM-guided SR

All SR procedures after FNAM were performed at a separate visit after the initial mapping procedure. If FNAM demonstrates 6 or more positive sites per testis and at least three positive contiguous sites per testis (i.e., diffuse hypospermatogenesis), a targeted testicular sperm aspiration (TESA) is attempted. If inadequate tissue or sperm are obtained with TESA, the procedure is converted to a conventional open testicular sperm extraction (TESE) in the same setting. These procedures are typically performed under local anesthesia, excepting patient preference.

If FNAM shows significant heterogeneity or focal hypospermatogenesis with less than 6 positive sites per testis (i.e., focal hypospermatogenesis), a microTESE is offered. If the FNAM is positive for spermatozoa bilaterally, then the side with higher number of FNA-positive sites is explored first. While the procedure begins identically to upfront microTESE, sites found to show mature spermatozoa on FNAM are targeted based upon positive locations from the FNAM. If sperm are found, adequate tissue is harvested from the site for IVF/ICSI, as verified in real-time by the same andrology team. If no sperm are found at the FNA-positive sites, a systematic exploration of all areas is performed as described for standard microTESE. If no sperm are found, the contralateral testis is explored regardless of FNAM positivity.

FNAM is performed and results used to inform patient counseling prior to starting an IVF cycle. Preemptive oocyte retrieval is performed prior to microTESE procedures with the intention of ‘fresh’ sperm utilization.

Statistical analysis

Data was maintained in a secure, encrypted file. Microsoft Excel (Microsoft Corporation) was utilized for data analysis. SR, pregnancy, and live-birth rates for the two cohorts were determined based upon an intention-to-treat analysis. Chi-Square analysis and Student’s t-test were used as appropriate.

Results

Patient characteristics

Ninety-six men with NOA met inclusion criteria. Of these, 60 men underwent FNAM, while 36 men underwent upfront microTESE (Table 1). The median patient age was 34 years in both cohorts, while the partner age was 32 years in the FNAM cohort and 33 years in the microTESE cohort. Patients with YCMD AZFc (n=4) were provided the option of either FNAM or upfront microTESE. There were no significant differences between the two cohorts. Table 1 provides patient characteristics, SR, pregnancy, and live-birth rates for both cohorts.

FNAM findings

Fifty-nine of 60 men demonstrated adequate tissue sampling for interpretation. The clinical pathway and outcomes of these men are shown graphically in Figure 1. One man with a history of gonadotoxic systemic chemotherapy and radiation was noted to have dense fibrosis, resulting in specimen inadequacy and early termination of the procedure. This patient underwent a microTESE as a separate, subsequent procedure.

Figure 1 FNAM clinical pathway. FNAM, fine needle aspiration mapping; TESA/E, testicular sperm aspiration/extraction; SR, sperm retrieval; IVF, in vitro fertilization.

Overall, 34 men (56.7%) were noted to have mature spermatozoa on FNAM (Table 2). Of these, 21 men demonstrated diffuse spermatogenesis (≥6 FNA-positive sites per testis); 14 men demonstrated this pattern bilaterally. The remaining 13 men demonstrated focal spermatogenesis—unilateral in 6 and bilateral in 9. Of the 25 men (41.7%) that did not have mature spermatozoa present on FNAM cytologic evaluation revealed germ cell aplasia in 21 men and MA in 4 men.

FNAM-guided SR

Thirty-one of 34 men proceeded with FNAM-guided SR (Table 3), while 3 men declined further intervention. The decision to forego SR was not based upon the findings of FNAM therefore they were removed from the outcomes analysis. Overall, 30 of 31 men who had spermatogenesis on FNAM and underwent SR were successful. Twenty of 21 men with diffuse spermatogenesis underwent FNAM-guided testicular sperm aspiration or extraction (TESA/E), while 1 man declined SR. Seventeen of 20 TESA/E procedures were performed under local anesthesia, and all 20 procedures were successful in the retrieval of sperm deemed adequate for IVF/ICSI (Table 3).

Of the 13 men with focal hypospermatogenesis, 11 underwent an FNA-guided microTESE, and 10 of 11 (91%) were successful in SR adequate for IVF/ICSI. Two men with focal hypospermatogenesis declined SR via microTESE. Of the 11 microTESE patients, 7 (64%) underwent a unilateral procedure. The patient with the aborted FNAM procedure underwent a bilateral microTESE with success resulting in adequate sperm for IVF/ICSI (Table 3).

No patients with a negative FNAM underwent attempted SR. Table 4 outlines partner oocyte management of both cohorts. Notably, no female partners in the FNAM-guided TESA/E sub-cohort underwent preemptive oocyte retrieval. All female partners in the FNAM-guided microTESE sub-cohort underwent preemptive oocyte retrieval.

Upfront microTESE findings

Of the 30 men that underwent upfront microTESE, 24 female partners had preemptive oocyte retrieval (Table 4). Seventeen of 30 men (57%) had a successful microTESE with SR that was adequate for IVF/ICSI (Table 1). In total, 16 men (44%) underwent unilateral microTESE, while 14 men underwent bilateral microTESE.

Overall, there was no statistical difference in SR rates (P=0.65) between the FNAM and upfront microTESE cohorts (Table 1).

IVF and pregnancy outcomes

Among the FNAM cohort, 30 of 31 men had successful SR (96.8%) used for IVF/ICSI, resulting in 24 clinical pregnancies for an overall pregnancy rate of 42.1% (24/57). All 31 men who underwent attempt at SR were included in the analysis of IVF and pregnancy outcomes. Seventeen of the 24 clinical pregnancies occurred in the TESA/E sub-cohort, while 7 pregnancies occurred in the microTESE sub-cohort. Notably, prior or simultaneous oocyte retrieval was avoided in all patients undergoing TESA/E. Overall, 21 live births were recorded (36.8%).

In the upfront microTESE cohort, all 17 couples with successful microTESE proceeded to IVF/ICSI with partner sperm. Of these, 11 clinical pregnancies were recorded. In those with unsuccessful microTESE, 6 couples declined IVF with donor sperm despite prior oocyte cryopreservation, while 3 couples proceeded with IVF using donor sperm. The overall pregnancy rate using partner sperm in the upfront microTESE cohort was 36.6%. All 11 clinical pregnancies resulted in live births (Table 4).

Finally, there was no statistical difference in pregnancy rates (P=0.76) and live-birth rates (0.75) between the FNAM and upfront microTESE cohorts (Table 1).

Discussion

Key findings

In the absence of reliable non-invasive predictors of SR success, couples with NOA face high levels of uncertainty. This uncertainty is further paired with high costs of treatment—both financial and emotional. With the aim to demonstrate a reliable predictor of SR success, our study finds that men with NOA who undergo initial diagnostic FNAM versus upfront microTESE have equal rates of successful SR, pregnancy, and live birth rates. Furthermore, FNAM group underwent fewer invasive procedures, and uncertainty throughout the process. These findings are generalizable to men with NOA.

A critical takeaway from our study is the power FNAM can provide reproductive urologists to individualize SR, allowing the use of less costly and less invasive techniques while enhancing confidence in success. We found that 21 of 60 patients (35%) demonstrated diffuse hypospermatogenesis, and underwent a minimally invasive TESA/E. Predictably, all 20 men underwent SR resulting in cryopreserved sperm sufficient for IVF/ICSI without preemptive oocyte retrieval for their partners. Arguably, this subcohort of men would have been successful in an upfront conventional TESE, as studies attribute a success rate of 16–45% with conventional TESE in NOA (9). In our study, we also identified 13 additional patients with focal hypospermatogenesis, 11 of whom underwent an FNAM-guided microTESE with >90% SR success. This subcohort of men likely represents those men that would be potentially missed via an upfront conventional TESE, but would ultimately have successful retrieval via microTESE. We do acknowledge that in many men this represents and additional procedure and additional time to the couple’s fertility journey. We believe that the predictability and potential cost savings this pathway provides enough value to justify it, however some couples may prefer to proceed directly to attempts at SR.

While the overall rate of complications for SR techniques is low, there appear to be some potential complications of microTESE including a transient decrease in serum testosterone (23). More serious complications of hematoma and testicular atrophy are possible but rare (9). In comparison the safety and non-invasiveness of FNAM is well established in the literature. In a large patient series there were no clinical or surgical complications (19). TESA has also been studied and found to be safe and is not associated with new-onset hypogonadism (24). FNAM allows for some patient to undergo TESA instead of microTESE with reliable results, which may prevent new hypogonadism in these men.

Finally, the 26 patients (42.3%) with no mature spermatozoa on FNAM avoided a costly and invasive procedure for themselves and their female partners did not undergo preemptive oocyte—a procedure carrying further risk and cost. While we do know that FNAM carries a high specificity through cytologic confirmation of presence or absence of spermatozoa, we believe that it is also highly sensitive based upon the comparable SR rates between the two SR pathways shown in this study. There is no published data that investigates the success of microTESE after FNAM shows no mature spermatozoa. Owing to the high sensitivity of the FNAM procedure, patients are counseled that the utility of attempts at SR is poor. There is published data of FNAM after failed microTESE with FNAM identifying sperm in 24/82 (29.3%) of such men (25). This provides some additional evidence that FNAM is highly sensitive and may even identify sperm in men who have negative upfront microTESE.

There is likely to be a difference in cost between these management pathways. While a formal comparison of cost was outside of the scope of the current study for the patients who avoided microTESE and preemptive oocyte retrieval they will certainly have reduced costs. Whether the addition of FNAM and the subsequent changes in choosing to undergo SR and by which method will have an effect on overall patient or system cost is unknown and an opportunity for further work. Coupling outcome predictability with cost-effectiveness is vital for the future of reproductive medicine, as we increase access for all couples experiencing infertility regardless of financial status or insurance coverage. Therefore, utilization of FNAM-guided SR in NOA is an appealing alternative with comparable outcomes to upfront microTESE.

Strengths and limitations

There are several limitations to this study. Due to the retrospective and non-randomized nature of this study, it is vulnerable to bias. Foremost, selection bias may be present in a reproductive urologist recommending FNAM or microTESE. The three reproductive urologists involved in this study followed one of the two management pathways based upon their training and preference and the choice of pathway was not based upon patient characteristics. The exception to this was the management of Klinefelter’s syndrome, which all providers believed was best managed with upfront microTESE. An inherent limitation to this study is that no patients who had FNAM with no mature spermatozoa underwent subsequent microTESE. Literature does exist evaluating the success of FNAM following failed microTESE, Despite the lack of randomization, this is the first study to present data on alternative management pathways for NOA in a consistent institutional and laboratory setting, which is critically absent in reproductive urology literature.

Conclusions

We present a non-randomized cohort study comparing FNAM-guided SR to upfront microTESE in men with NOA that demonstrates comparable SR, pregnancy, and live-birth rates. As the first of its kind, this study also highlights a high degree of success in SR following FNAM. FNAM is an excellent alternative to upfront microTESE that empowers reproductive urologists with a reliable tool for counseling and management.

Acknowledgments

Funding: None.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-24-362/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-24-362/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-24-362/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-24-362/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was conducted with approval from the University of Washington Institutional Review Board (No. 00004466) and informed consent was waived due to its retrospective nature.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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