The power of the map: testicular mapping to guide surgical sperm retrieval in patients with non-obstructive azoospermia

Introduction

Non-obstructive azoospermia (NOA) is defined as the absence of sperm in the ejaculate without an identifiable obstructive cause (1). Patients with this clinical entity face a diagnostic dilemma: they are uncertain if they have sperm, and if they do, what is the optimal method to extract the sperm for use with assisted reproductive technologies. Acquiring sperm via testicular sperm aspiration (TESA) or through an incision via testicular sperm extraction (TESE) have been shown to be effective, minimally invasive approaches (2). The addition of an operative microscopic via microscopic testicular sperm extraction (mTESE) has been shown to further increase the success rate of acquiring sperm (3). However, these different modalities each pose an escalation in care with additional financial, biologic, and emotional costs to patients. Published success rates of mTESE in expert hands are around 60% (3,4). For couples to use rare surgically retrieved sperm, upfront oocyte retrieval and/or cryopreservation may be required. The invasiveness and cost of oocyte retrieval can be deterring to couples especially when considering the 40% failure rate of mTESE. A less invasive strategy that allows for upfront sperm retrieval and cryopreservation for appropriately selected patients with NOA is needed. Diagnostic uncertainty led to the development of fine needle aspiration mapping (FNAM) of the testicle (5). FNAM involves systematically aspirating multiple sites of each testicle with a fine-gauged needle to cytologically identify sperm (5). Briefly, a 24-gauge needle is used to mechanically aspirate macroscopic tubules, which are then cut and smeared on a slide. The systematic sampling has diagnostic utility and may help guide subsequent sperm extraction attempts when compared with a single testicular biopsy site alone. FNAM is highly sensitive, with prior work demonstrating that FNAM after a failed mTESE may result in the subsequent identification of sperm, thereby guiding retrieval (6). FNAM may allow clinicians to better understand the distribution of sperm within the testicles prior to extraction efforts, and therefore inform clinical decision making for individual patients (5-9).

Other clinical metrics have also shown diagnostic utility. A follicle stimulating hormone (FSH) level of 7.6 U/L has been reported to differentiate patients with obstructive versus NOA (1). Subsequent studies have analyzed FSH levels which portend successful sperm extraction and found cutoffs anywhere ranging from 7 to 13.6 U/L (10-13). Some of this variability may be related to how sperm presence is defined, ranging from testicular biopsy to mTESE. While a single laboratory test in isolation may be insufficient to fully prognosticate a patient’s fertility status, a definitive investigation of clinically impactful FSH levels is important and lacking from the literature.

We hypothesize that FNAM can provide practical diagnostic value by de-escalating sperm extraction technique selection without sacrificing success rate, even in patients with poor clinical predictors of sperm presence. We also hypothesize that clinically impactful FSH levels will exceed contemporary cut-off values when precise FNAM cytologic findings are used to determine sperm presence or absence. We present this article in accordance with the STROBE reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-24-498/rc).

Methods

Subject population

With institutional review board (IRB) approval, we performed a retrospective review of all patients 18 years and older who underwent FNAM at our institution between January 2008 and August 2023. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the IRB of University of Washington (No. 00018380) and individual consent for this retrospective analysis was waived.

FNAM

Fine needle aspiration testicular mapping was performed by three different surgeons, with all surgeons using the same technique. Using local anesthesia, a total of 12 systematic samples were obtained from each available testicle, using a 23-gauge needle and suction-cutting technique (5,14). Tissue samples were smeared on a slide, fixed in methanol, stained with Quick III staining solution, and cover-slipped for analysis as previously described (5). A schematic of laboratory workflow is shown in Figure 1. In some cases of testicular fibrosis, small testicular size, or patient intolerance, fewer samples were taken. If a patient had two testicles, both were assessed via fine needle aspiration. In the cases of solitary testicles (for any reason), severely atrophic or hypoplastic testicles, undescended testicle, or severe discomfort, a single testicle was sampled.

Figure 1 Overview of procedural workflow for fine needle aspiration mapping of the testicles.

FNAM slides were evaluated by one or two individuals with expertise in identification of testis cells. The adequacy of tissue for evaluation was assessed by estimates of the number of Sertoli cells present. Sertoli cells were quantified on a logarithmic scale for each slide (site) with ≥1,000 per slide considered normal for the usual length of tubule aspirated. Normal spermatogenesis (NS) was defined as the majority of sites containing over 25 sperm per slide. Diffuse hypospermatogenesis (HS) was between 1 and 25 sperm per slide. Focal HS was diagnosed when fewer than half the sampled sites on a testicle had any sperm, and those sites had between 1 and 25 or more sperm per slide. If one testicle had focal HS and the other had diffuse, the patient would be given the diagnosis of diffuse HS. HS is sub-stratified between focal and diffuse by the fertility lab to capture additional testicular topographic information and aid in clinical decision making. Maturation arrest (MA) was defined by the presence of immature germ cells and no testicular spermatozoa. Germ cell aplasia (GCA) was defined as the presence of only Sertoli cells with no germ cells or sperm present. Images of each cytologic diagnosis are shown in Figure S1.

Clinical data

Clinical data were abstracted from records in an electronic medical record system, stored, and managed in REDCap electronic data capture tools hosted at the University of Washington (15,16). Body mass index (BMI) was calculated as a continuous value and categorized into commonly accepted cut-offs.

Statistical analysis

Statistical analysis was performed using Microsoft Excel for Microsoft 365 Version 2311 (Redmond, WA, USA) and GraphPad Prism version 10.1.0 (Boston, MA, USA). A P value less than 0.05 was determined to be statistically significant. We used unpaired t-tests and chi squared tests to compare groups for continuous and categorical variables, respectively. We conducted one-way analysis of variance (ANOVA) analysis with subsequent multiple comparisons between detailed FNAM diagnosis using the Dunn-Sidak correction.

Results

Two hundred twenty consecutive patients over the age of 18 underwent FNAM at our institution with 419 total testicles assessed. Median follow up was 78 months. Testicular sperm was identified in 84 individuals while no sperm was identified in 136 patients. Twenty-one patients in our cohort had only a single side sampled due to solitary testis or for other reasons listed. Of the remaining patients who underwent bilateral FNAM, 176 (88%) had identical FNA histologic diagnoses between the two testicles while 23 (12%) had discordance. There was no statistically significant difference in sperm detection in right versus left testicles in those patients where both were sampled. When considering all attempted collection, the adequacy rate of FNAM sampling was 96.5% as defined by the presence of Sertoli cells. Fewer than 12 samples were taken from an individual testicle in 4.5% of cases due to fibrosis, size, or patient intolerance. Of the 220 patients, 212 had semen analysis available for detailed review. Semen analysis always included evaluation of concentrated (centrifuged) semen when no sperm were found in 20–100 hpf of neat semen and were analyzed in accordance with the World Health Organization (WHO) protocol. In the cohort with sperm found on FNAM, 73 (90%) had azoospermia whereas 8 (10%) had cryptozoospermia (sperm only found in concentrated semen). In the cohort with no sperm found on FNAM, 125 (95%) had azoospermia and 6 (5%) had cryptozoospermia. Azoospermia was confirmed on two separate semen analyses. Three patients had failed mTESE procedures prior to FNAM, all of which had no sperm on FNAM.

The average age for the cohort was 43 years old. There was no statistically significant difference in age between the groups that did or did not have sperm found on FNAM (Table 1). There was no difference in BMI between the two groups. Average testicular size based on physical exam was statistically larger in patients with sperm identified on FNA mapping, with an absolute difference of 1.25 mL (Table 1). Varicoceles were detected in 12% of each group. Medication use at the time of FNA mapping was similar between the groups. Marijuana usage was statistically higher in the group with sperm detected on FNA map: 13% of patients with sperm found on FNA map reporting usage of marijuana on a regular basis compared to 4% in the group with no sperm found on FNA map. Y chromosome microdeletions were seen in 7 patients (13%) with no sperm on their FNA map and no patients with sperm on FNAM. Of these 7 patients, 3 had AZFc, 2 had AZFb, 1 had AZFb+c, and 1 had deletion on the short arm of the y chromosome not mapped to the AZF regions. These patients all had normal karyotypes. One patient had an abnormal karyotype of 47 XYY with isodicentric Y and did not have sperm on FNAM. Of the 220 patients who underwent FNAM, 4 had prior testis biopsy and 7 had prior attempts at therapeutic extraction with 2 being successful.

Two adverse events occurred following FNAM, both of which required no additional intervention. One patient reported “bruising and pain” that lasted for one month, while another reported pain and had a small hematoma identified on ultrasound that also resolved without intervention.

Beyond the binary presence of sperm, the number of regions and amount of sperm and germ cells were quantified for each FNA map. Diagnoses were applied as described above by two fertility laboratory specialists. Of the 136 patients with no sperm, 102 had GCA, 32 had MA, and 2 had tubular fibrosis. Of the 84 patients with sperm, 33, 25, and 26 patients had NS, diffuse HS, and focal HS, respectively.

FSH levels were statistically higher in the group who did not have sperm on their FNA map (19.6 vs. 13.9 mU/mL) (Table 1). Using sub-stratification with diffuse and focal HS combined, we compared the hormone levels of all patients (Figure 2). Patients with HS had a higher average FSH of 18.4 mU/mL compared with 7.3 mU/mL in the group with NS (P<0.001). No significant difference in FSH level was seen between those with HS and those with GCA or MA. In men with FSH levels between 7.7 and 14.9 mU/mL, 36% were found to have sperm. In men with FSH exceeding 15 mU/mL, 28% were found to have sperm on FNAM. A significant difference was also seen in luteinizing hormone (LH) levels for patients with HS compared with NS, which was 7.9 and 4.5 mU/mL, respectively (P=0.01). While LH was higher in the group without sperm, testosterone and prolactin were not statistically different between the two groups (Figure 2).

Figure 2 Hormone levels stratified by fine needle aspiration diagnosis. (A) Median serum levels of FSH. (B) Median serum levels of testosterone. (C) Median serum levels of LH. (D) Median serum levels of prolactin. ns, not significant; *, P <0.05; ****, P<0.001. FSH, follicle stimulating hormone; GCA, germ cell aplasia; HS, hypospermatogenesis; LH, luteinizing hormone; MA, maturation arrest; NS, normal spermatogenesis.

Of the 84 patients with documented sperm on FNA mapping, 52 went on to TESE at our facility (Figure 3). Sperm extraction technique selection was determined largely based on FNAM results within our clinical algorithm. Patients with NS or diffuse HS underwent TESA or TESE while patients with focal HS underwent mTESE. Specifically, 30 patients underwent TESA, 18 mTESE, and 4 conventional TESE. Of note, all TESE and TESA procedures were performed in the clinic with administration of local anesthesia. Sperm extraction was unilateral in 92% of cases. Successful sperm retrieval for in vitro fertilization (IVF) was achieved in all procedures, with the choice of surgical sperm retrieval (SSR) technique guided by FNAM results. Notably, 6 of the 18 mTESE procedures specifically targeted testicular areas identified through FNAM guidance. There were 11 conceptions and 7 live births documented, however conception data was not systematically queried from patients as part of routine clinical practice. The 136 patients with no sperm identified on FNAM were offered mTESE however were counseled of a low chance of success. No patients without sperm on their FNAM elected to undergo SSR.

Figure 3 Surgical sperm extraction rate in patients who had sperm present on fine needle aspiration testicular mapping (N=52). mTESE, microscopic testicular sperm extraction; TESE, testicular sperm extraction; TESA, testicular sperm aspiration.

All 52 patients who underwent TESE had available FSH data. Surgical sperm extraction technique stratified by FSH level is summarized in Table 2. Sperm extraction technique selection was guided by FNA results and not FSH level. In those with low levels of FSH based on American Urological Association (AUA) guidelines (less than 7.6 mU/mL), only 9% required mTESE. In those with slightly higher levels of FSH (7.7–14.9 mU/mL), 40% required mTESE. In those with significantly elevated FSH levels (greater than 15 mU/mL), 60% required mTESE, while 40% achieved successful sperm retrieval with office-based techniques.

In the 35 patients for whom sperm was identified with a complete 24-point FNA map, we performed sperm localization interrogation using chi squared analysis. There was no statistically significant area in which sperm was more likely to be identified (Figure S2).

Discussion

Our study serves as a large, contemporary exploration of the pragmatic utility of FNAM. We demonstrate that FNAM can be used to accurately stratify patients to less invasive TESE techniques despite unfavorable clinical characteristics. Specifically, 65% of patients who underwent SSR were able to avoid mTESE in favor of office-based procedures. FNAM enabled precise targeting of our sperm retrieval approach, resulting in a 100% success rate with the chosen extraction technique. This may provide couples undergoing simultaneous egg and sperm retrieval with additional clinical clarity and decrease the likelihood of freezing unfertilized oocytes or needing to use donor sperm. This study is the first to assess clinical outcomes and hormonal variables after substratifying to a more precise cytopathologic diagnosis such as HS. Moreover, some patients met clinical criteria for NOA yet were found to have NS on FNAM. The diagnostic information from FNAM identified these patients with occult obstructive azoospermia, enabling use of less invasive SSR.

We demonstrate that precise cytological diagnosis through FNAM effectively guides sperm extraction technique. De-escalation of SSR technique may help avoid the financial burden, longer procedure time, requirement of a microsurgeon, and general anesthesia of a mTESE (4,17,18). The added diagnostic value of FNAM may also help engage patients in enhanced shared decision-making prior to SSR.

Importantly, we also show that while FSH is a predictor of sperm detection, high levels should not deter patients or clinicians from attempts at SSR, given the average value in our HS cohort was 18.4 U/L. Previous studies have suggested cut points ranging from 7.6 to 13.6 U/L which would not capture all men with HS (10-12). Among patients with sperm identified on FNA map and FSH levels exceeding 15 U/L, 40% achieved successful sperm extraction with an office-based technique guided by FNA map results. In our study, FSH levels do not statistically distinguish patients with HS from those with no sperm on their FNA map. Our findings suggest that FNAM can optimize the selection of sperm extraction techniques for men with elevated FSH levels.

The current cohort demonstrated a very low complication rate after FNAM, with only 2 patients experiencing adverse events, both of which were mild and abated with observation. In contrast, extraction modalities such as multiple TESE sites or mTESE have known complications such as infection, subsequent hypogonadism and testicular atrophy (19-21). While FNAM does not obviate the need for future sperm extraction, FNAM itself does not appear to cause significant morbidity. Indeed, one drawback of FNAM is that it is an additional diagnostic test that poses opportunity and financial cost in a process with patients undergoing large amounts of testing.

Testicular size was a predictor of finding sperm on FNAM; however, the average size of 16 cc in the sperm-positive group was clinically similar to the 14 cc average in the sperm-negative group. While we did observe a difference in LH between the patients with and without sperm on FNAM, it is unclear what clinical significance this has, as testosterone levels were not significantly different. Additionally, marijuana use was counterintuitively reported at higher rates in the group that had sperm identified. This may be a product of the real-world nature of our results and low amount of marijuana usage overall.

Our FNAM technique involved attempting to sample 12 sites per testicle, however the literature does note variance in sampling approaches ranging from 4 to 18 sites per testicle (22). Sperm detection rate does increase with additional sampling, ranging from 47% in those with 7 sites sampled up to 60% in those with 18 sites sampled (22). We selected 12 as we believe this to be an optimized middle ground. However, it is possible that with additional or fewer sample sites, the FNAM diagnoses would change in a minority of patients.

Other studies attempting to identify areas of the testicle with greater likelihood of sperm detection have found mixed results (6,7). Turek et al. [2000] found no correlation while Jarvis et al. [2019] showed a proclivity for the lateral pole of the right testis and the superior aspect of the left testis (6,7). Our study supports the findings of Turek et al. [2000] which found that there are no anatomic areas of greater sperm production in patients seeking assisted reproductive technologies. Jarvis et al. [2019] found areas of increased spermatogenesis, however their patient population had all undergone failed mTESE prior to FNAM. It is possible that the prior intervention altered the testicular architecture or that the subpopulation selected by failing mTESE led in this statistical finding. Importantly our topographic analysis could only be performed in patients with material isolated from all 24 sites. In patients with sperm on their FNA map, 35 (42%) had successful collection from all 24 sites, so a larger sample size may be required to fully evaluate this phenomenon.

This study has several limitations including its retrospective nature. Patient follow up was not systematically assessed; however, all subsequent clinical encounters at our institution were retrospectively accessed. While all patients with sperm identified by FNAM had successful sperm retrieval, no patients with negative FNAM underwent surgical sperm extraction. Therefore, the likelihood of successful mTESE despite a negative FNAM is unknown. Additionally, not all patients with sperm on their FNA map went on to surgical sperm extraction at the time of chart review. The reason these patients did not pursue SSR was not identified on chart review. While the diagnostic value of a negative FNAM is not clear, a positive FNAM portends successful sperm retrieval with the least invasive approach.

Conclusions

FNAM allows for sperm identification in patients with NOA who were historically thought to be poor candidates for sperm extraction. FNAM allows for de-escalation of extraction methodology used, while providing patients and providers more diagnostic certainty. Elevated FSH levels should not preclude attempts at surgical sperm extraction, and patients with significantly elevated levels may de-escalate their extraction technique based on the FNA map. Future investigations into FNAM outcomes are warranted. Qualitative studies examining how the patient experience is impacted by the diagnostic information obtained by FNAM may help guide patient counselling. Studies directly comparing SSR rates guided by FNAM versus upfront mTESE would help clarify FNAM’s therapeutic utility.

Acknowledgments

We would like to acknowledge the work of the Male Fertility Lab in supporting the technical aspects of this work. A portion of these data were presented at the American Society for Reproductive Medicine 2024 annual meeting.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-24-498/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-24-498/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-24-498/prf

Funding: There was no specific funding to report. The REDCap instance used is supported by the Institute of Translational Health Sciences, which is funded by the National Center for Advancing Translational Sciences of the National Institutes of Health under award number UL1TR002319.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-24-498/coif). T.J.W. serves as a consultant for the Progyny company which focuses on providing solutions for male infertility. The other 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 approved by the institutional review board of University of Washington (No. 00018380) and individual consent for this retrospective analysis was waived.

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/.

References

1. Schoor RA, Elhanbly S, Niederberger CS, et al. The role of testicular biopsy in the modern management of male infertility. J Urol 2002;167:197-200. [Crossref] [PubMed]
2. Mulhall JP, Burgess CM, Cunningham D, et al. Presence of mature sperm in testicular parenchyma of men with nonobstructive azoospermia: prevalence and predictive factors. Urology 1997;49:91-5; discussion 95-6. [Crossref] [PubMed]
3. Schlegel PN. Testicular sperm extraction: microdissection improves sperm yield with minimal tissue excision. Hum Reprod 1999;14:131-5. [Crossref] [PubMed]
4. Ramasamy R, Padilla WO, Osterberg EC, et al. A comparison of models for predicting sperm retrieval before microdissection testicular sperm extraction in men with nonobstructive azoospermia. J Urol 2013;189:638-42. [Crossref] [PubMed]
5. Turek PJ, Cha I, Ljung BM. Systematic fine-needle aspiration of the testis: correlation to biopsy and results of organ "mapping" for mature sperm in azoospermic men. Urology 1997;49:743-8. [Crossref] [PubMed]
6. Jarvis S, Yee HK, Thomas N, et al. Sperm fine-needle aspiration (FNA) mapping after failed microdissection testicular sperm extraction (TESE): location and patterns of found sperm. Asian J Androl 2019;21:50-5. [Crossref] [PubMed]
7. Turek PJ, Ljung BM, Cha I, et al. Diagnostic findings from testis fine needle aspiration mapping in obstructed and nonobstructed azoospermic men. J Urol 2000;163:1709-16. [Crossref] [PubMed]
8. Lewin A, Reubinoff B, Porat-Katz A, et al. Testicular fine needle aspiration: the alternative method for sperm retrieval in non-obstructive azoospermia. Hum Reprod 1999;14:1785-90. [Crossref] [PubMed]
9. Meng MV, Cha I, Ljung BM, et al. Relationship between classic histological pattern and sperm findings on fine needle aspiration map in infertile men. Hum Reprod 2000;15:1973-7. [Crossref] [PubMed]
10. Mourad WA, Tulbah A, Merdad T, et al. Fine-needle aspiration of the testis in azoospermic men: the value of measuring serum follicle stimulating hormone and testicular size. Diagn Cytopathol 2005;32:185-8. [Crossref] [PubMed]
11. Tüttelmann F, Werny F, Cooper TG, et al. Clinical experience with azoospermia: aetiology and chances for spermatozoa detection upon biopsy. Int J Androl 2011;34:291-8. [Crossref] [PubMed]
12. Bromage SJ, Falconer DA, Lieberman BA, et al. Sperm retrieval rates in subgroups of primary azoospermic males. Eur Urol 2007;51:534-9; discussion 539-40. [Crossref] [PubMed]
13. Jahromi BN, Zeyghami S, Parsanezhad ME, et al. Determining an optimal cut-off value for follicle-stimulating hormone to predict microsurgical testicular sperm extraction outcome in patients with non-obstructive azoospermia. Arch Endocrinol Metab 2020;64:165-70. [Crossref] [PubMed]
14. Ljung BM. Techniques of aspiration and smear 317 preparation. In: Koss LG, Woyke S, Olszewski W, editors. Aspiration Biopsy. Cytologic Interpretation and Histologic Bases. 2nd ed. New York: Igaku-Shoin; 1992.
15. Harris PA, Taylor R, Minor BLThe REDCap consortium, et al. Building an international community of software platform partners. J Biomed Inform 2019;95:103208. [Crossref] [PubMed]
16. Harris PA, Taylor R, Thielke R, et al. Research electronic data capture (REDCap)--a metadata-driven methodology and workflow process for providing translational research informatics support. J Biomed Inform 2009;42:377-81. [Crossref] [PubMed]
17. Patel SR, Park B, Reddy A, et al. Testicular Core Extraction: Important Technique for Determining Sperm Retrieval Method in Non-obstructive Azoospermia. Urology 2023;173:87-91. [Crossref] [PubMed]
18. Jensen CF, Ohl DA, Hiner MR, et al. Multiple needle-pass percutaneous testicular sperm aspiration as first-line treatment in azoospermic men. Andrology 2016;4:257-62. [Crossref] [PubMed]
19. Ramasamy R, Yagan N, Schlegel PN. Structural and functional changes to the testis after conventional versus microdissection testicular sperm extraction. Urology 2005;65:1190-4. [Crossref] [PubMed]
20. Schlegel PN, Su LM. Physiological consequences of testicular sperm extraction. Hum Reprod 1997;12:1688-92. [Crossref] [PubMed]
21. Ishikawa T, Yamaguchi K, Chiba K, et al. Serum hormones in patients with nonobstructive azoospermia after microdissection testicular sperm extraction. J Urol 2009;182:1495-9. [Crossref] [PubMed]
22. Beliveau ME, Turek PJ. The value of testicular 'mapping' in men with non-obstructive azoospermia. Asian J Androl 2011;13:225-30. [Crossref] [PubMed]