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Outcomes of microdissection testicular sperm extraction in men with nonobstructive azoospermia due to maturation arrest
ORIGINAL ARTICLE: ANDROLOGY
Outcomes of microdissection testicular sperm extraction in men with nonobstructive azoospermia due to maturation arrest Aaron M. Bernie, M.D., M.P.H.,a Kalee Shah, B.A.,a Joshua A. Halpern, M.D.,a Jason Scovell, B.A.,b Ranjith Ramasamy, M.D.,b Brian Robinson, M.D.,c and Peter N. Schlegel, M.D.a a Department of Urology, Weill Cornell Medical College, New York, New York; b Baylor College of Medicine, Houston, Texas; and c Department of Pathology & Laboratory Medicine, Weill Cornell Medical College, New York, New York
Objective: To evaluate sperm retrieval in men with nonobstructive azoospermia and maturation arrest (MA) undergoing microdissection testicular sperm extraction (micro-TESE). Design: Retrospective chart review. Setting: Tertiary referral center. Patient(s): Men with nonobstructive azoospermia and MA who underwent micro-TESE. Intervention(s): Microdissection TESE. Main Outcome Measure(s): Sperm retrieval rate (SRR). Result(s): A total of 211 patients (13%) had a histologic finding of MA at the most advanced level. The overall SRR was 52%. A total of 146 patients were classified as having early MA (arrest at the primary spermatocyte stage), and 65 as having late MA (early spermatid stage). The SRR in men with early, vs. late, MA was 40% vs. 78%. Of the 211 men with MA, 51 had diffuse MA (100% of tubules showed MA). The SRR was significantly lower in men with diffuse vs. focal MA (35% vs. 57%). On multivariable analysis, late MA and higher follicle-stimulating hormone levels were positively associated with successful sperm retrieval. Conclusion(s): Sperm were successfully identified in up to one half of the men with MA after micro-TESE. Among men with MA, late MA seems to be the best predictor of successful sperm retrieval with micro-TESE. (Fertil SterilÒ Use your smartphone 2015;-:-–-. Ó2015 by American Society for Reproductive Medicine.) to scan this QR code Key Words: Microdissection testicular sperm extraction, maturation arrest, nonobstructive and connect to the azoospermia Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/berniea-micro-tese-maturation-arrest/
INTRODUCTION Nonobstructive azoospermia (NOA), or testicular failure, affects 1% of males and 10% of those who seek fertility assistance (1). Three histopathological patterns are typically described in the biopsies of men who have NOA: reduced spermatogenesis with all germ cell types present (hypospermato-
genesis); premature arrest of spermatogenesis (maturation arrest [MA]); and complete absence of germ cells (Sertoli-cell-only syndrome) (2). Limited biopsies may fail to identify focal areas of sperm production that may otherwise be found with a detailed micro– testicular sperm extraction (TESE) search. Clinically, the histologic classi-
Received April 7, 2015; revised May 13, 2015; accepted May 29, 2015. A.M.B. has nothing to disclose. K.S. has nothing to disclose. J.A.H. has nothing to disclose. J.S. has nothing to disclose. R.R. has nothing to disclose. B.R. has nothing to disclose. P.N.S. has nothing to disclose. Reprint requests: Peter N. Schlegel, M.D., Department of Urology, Brady Urologic Health Center, 525 East 68th St, Starr Pavilion, 9th Floor, New York, New York 10065 (E-mail: [email protected]. edu). Fertility and Sterility® Vol. -, No. -, - 2015 0015-0282/$36.00 Copyright ©2015 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2015.05.037 VOL. - NO. - / - 2015
discussion forum for this article now.*
* Download a free QR code scanner by searching for “QR scanner” in your smartphone’s app store or app marketplace.
fication on a diagnostic biopsy can provide value in predicting the chance of success with TESE. For instance, the average sperm retrieval rate (SRR) is 94% in patients with hypospermatogenesis, compared with 40% in patients with Sertoli-cell-only syndrome, even with micro-TESE (3, 4). Maturation arrest is defined as an absence of mature spermatozoa as a result of an early arrest in germ cell development in the seminiferous tubules. It is further delineated into early and late MA, based on the mostmature cell type noted on histology. In early MA, spermatogenic arrest occurs at the spermatogonia or spermatocyte stage; in late MA, arrest occurs at the 1
ORIGINAL ARTICLE: ANDROLOGY spermatid stage (5–7). Late MA is distinguished from hypospermatogenesis in that no condensed, oval spermatids are seen in late MA. The underlying genetic causes that result in the phenotypes of early vs. late MA remain undetermined. On biopsy, MA can also vary in its heterogeneity, from focal to diffuse, further complicating prediction of SRR with TESE. In men with MA, studies show a highly variable SRR of 23%–50% (6, 7). Both Tsai et al. (6) and Weedin et al. (7) found that, compared with men with late MA, those diagnosed with early MA had a decreased probability of successful sperm retrieval. Due to the limited number of cases in prior publications, histopathology could not be used to predict successful TESE. Therefore, we present a series of patients with NOA who underwent micro-TESE, performed by a single surgeon at our center, and were diagnosed with MA. We classified men with MA as either early or late, and focal or diffuse, and evaluated sperm SRR.
METHODS After receiving approval from the Weill Cornell Medical College Institutional Review Board, we retrospectively reviewed the charts of 1,686 consecutive patients who had NOA, confirmed by analysis of 2 centrifuged semen samples, from November 1995 through June 2014. All patients who were reviewed underwent micro-TESE at a single center, performed by a single surgeon. The NOA was confirmed via repeat semen analysis on the day of micro-TESE. Men with complete azoospermia factor (AZF)a or AZFb microdeletions were excluded from our analysis: no men with AZFa or AZFb underwent micro-TESE. Preoperatively identifiable factors, including age, follicle-stimulating hormone (FSH), total serum testosterone (T), testis volume on physical exam, presence of a varicocele, history of cryptorchidism, history of testicular cancer, prior diagnosis of Klinefelter syndrome, and Y microdeletion (AZFc) were analyzed. Men with a history of varicocele underwent varicocelectomy before micro-TESE. Micro-TESE
was performed as described previously (8). All men underwent initial unilateral micro-TESE, and if no sperm were retrieved, the contralateral testis was explored. Sperm retrieved were cryopreserved according to patient preference. Although some patients were referred to our center with a previous diagnostic biopsy or TESE, we did not perform routine biopsies because a diagnostic biopsy does not predict the presence of focal areas of spermatogenesis that microTESE can identify (9). Given the limited information available from previous outside biopsies, and the small number of men who had them performed, histologic data from outside biopsies were not included in this analysis. The pathologic information used in this study was from a single random intraoperative biopsy taken at the time of micro-TESE. Patients were considered to have MA if the most advanced pathologic pattern on either diagnostic or random intraoperative testis biopsy was consistent with MA. Biopsy slides were then re-reviewed by our uropathologist, who was blinded to the outcome of micro-TESE, and classified as either early or late MA. Early MA was defined as arrest of spermatogenesis at the primary spermatocyte stage (nuclei with filamentous chromatin, Fig. 1). Late MA was defined as arrest of spermatogenesis at the early spermatid phase (dark round nuclei, Fig. 1) and the absence of elongated spermatids/spermatozoa (elongated/oval nuclei). Although previous studies have looked at men who have MA (6, 7, 10), no well-defined criteria have been established for determining if a patient is considered to have diffuse or focal MA. Thus, we established a classification scheme intended to delineate the 2 groups. Given that all pathology was from a single intraoperative biopsy taken during microTESE, we felt that if a biopsy showed 100% MA, i.e., all tubules examined had MA, then those patients likely had only MA in the remainder of the testis. In contrast, if a portion of the biopsy revealed an alternate pathology (i.e., Sertolicell-only syndrome or hypospermatogenesis), we classified those men as having focal MA. Successful sperm retrieval depends on having an area of the testis where complete
FIGURE 1
Early (left) and late maturation arrest (right). Bernie. Micro-TESE in men with maturation arrest. Fertil Steril 2015.
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Fertility and Sterility® univariate analysis were included for multivariable binary logistic regression. All statistical analysis was performed using SPSS, version 22 (IBM).
TABLE 1 Early vs. late maturation arrest. Variable n Sperm retrieval rate Male age (y) Female age (y) Average testicular volume (cc) FSH (mU/mL) Testosterone (ng/dL) Y microdeletion Cryptorchidism Klinefelter Varicocele Cancer Hx
All MA patients
Early MA
Late MA
P value
211 52 36 32 10
146 40 36 32 10
65 78 37 32 10
< .0001 .53 .96 .84
21 408 12 23 0 68 3
22 400 13 18 1 66 5
20 426 9 34 0 71 0
.22 .33 .58 .024 .86 .64 .17
RESULTS Overall Characteristics
Note: Values are %, or median, unless otherwise indicated. Bernie. Micro-TESE in men with maturation arrest. Fertil Steril 2015.
spermatogenesis occurs. If the testis is uniformly abnormal in function, such as that seen with a testis that is 100% MA, then mature sperm are unlikely to be present. Therefore, in this study, patients were classified as having diffuse MA if 100% of the tubules revealed MA, and as having focal MA if <100% of the tubules revealed MA. We performed both univariate and multivariable binary logistic regression analysis to identify factors associated with and predictive of positive sperm retrieval during a micro-TESE. For univariate analysis, we included subject age, testicular volume, serum T, luteinizing hormone (LH), FSH, early vs. late MA, diffuse vs. focal MA, history of cryptorchidism, and history of Klinefelter syndrome. We tested for data normality using a quantile-quantile (Q-Q) plot, along with the Shapiro-Wilk test. Non-normally distributed continuous variables were normalized using a logistic transformation and include T, FSH, and LH. Factors that were significantly (P< .05) associated with sperm retrieval on the
A total of 211 patients (13% of the total micro-TESE cohort) were found to have a most advanced histologic finding of MA on pathologic analysis. The median age was 36 (7) years. Median testicular volume was 10 (5) cc. The overall SRR for the cohort was 52%, with a clinical pregnancy rate of 44%. Characteristics for all MA patients are included in Table 1.
Early vs. Late Maturation Arrest Of the 211 men, 146 patients (69%) were classified as having early MA, and 65 (31%) were classified as having late MA, on pathology review (Fig. 2). Table 1 presents the characteristic differences between early and late MA. The SRR was higher in men who had late, vs. early, MA (78% vs. 40%; P< .0001). Men who had late MA were more likely to have a history of cryptorchidism, compared with men who had early MA (34% vs. 23%, P¼ .024). No differences were found in male or female age, testicular volume, FSH or T, diagnosis of Y microdeletion or Klinefelter's syndrome, varicocele, or history of cancer in men with early vs. late MA.
Diffuse vs. Focal Maturation Arrest Of the 211 men who had MA, 51 (24%) had diffuse MA (Fig. 2). The SRR was significantly lower in men with diffuse MA, compared with men with focal MA (35% vs. 57%, P¼ .008). Men with diffuse MA additionally had larger testicular size (13 vs. 8.7 cc, P< .001) and lower FSH levels (14 vs. 25 mIU/mL, P< .001). Men with focal MA were more likely to have a history of cryptorchidism, compared with men with diffuse MA (30% vs. 10%, P¼ .004).
FIGURE 2
Breakdown of men who had NOA and MA, into early vs. late, and diffuse vs. focal, MA. MA ¼ maturation arrest; NOA ¼ nonobstructive azoospermia; SRR ¼ sperm retrieval rate. Bernie. Micro-TESE in men with maturation arrest. Fertil Steril 2015.
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ORIGINAL ARTICLE: ANDROLOGY Predictors of Successful Sperm Retrieval On univariate linear regression analysis, we found that higher FSH, late MA, focal MA, and a history of cryptorchidism were positively associated with successful sperm retrieval (P< .05) (Supplemental Table 1, available online). However, on multivariable analysis, only late MA and higher FSH were positively associated with successful sperm retrieval (P< .05) (Table 2).
DISCUSSION Men who have NOA and MA represent a challenging group of patients to treat. In particular, the decision to pursue microTESE, and the determination of potential for SRR, can be complex. Thus, identification of clinical characteristics that can help predict micro-TESE outcome is valuable for clinical management and preoperative counseling of these patients, particularly those who need a repeat procedure after previous failed sperm retrieval. Although we do not advocate routine preoperative biopsies for men with NOA, if a previous biopsy or pathology from attempted TESE are available, they can be used in counseling. In such cases, we recommend that pathology from prior procedures be closely re-reviewed by a dedicated genitourinary pathologist to identify focal/diffuse and early/late MA, as these are critical to predicting the success of subsequent micro-TESE. We evaluated sperm retrieval in men who had MA and found an overall SRR of 52%. Men who had late and focal MA were more likely to have successful sperm retrieval than were men who had early and diffuse MA, respectively. On a multivariate analysis, both elevated FSH and late MA were found to be predictive of successful sperm retrieval. We found a higher SRR rate in men who had late MA, compared with men who had early MA (6, 7). Men who had late MA in our series were almost twice as likely to have sperm retrieved, compared with men who had early MA (78% vs. 40%). Late MA on diagnostic biopsy clearly suggests that the more advanced the pattern seen on biopsy, the more likely there are to be areas of complete spermatogenesis elsewhere in the testicle. Interestingly, men with a history of cryptorchidism were more likely to have late MA vs. early MA in our series, and the subset of men with both cryptorchidism and late MA had the highest SRR of any subgroup. No previous
TABLE 2 Multivariable factors predictive of finding sperm on microscopic TESE. Factor FSH Early (reference) vs. late maturation arrest Diffuse (reference) vs. focal Orchiopexy
Odds ratio
95% CI
P value
2.978 0.169
(1.061, 8.356) (0.080, 0.357)
.038 < .001
1.732 1.106
(0.786, 3.818) (0.504, 2.428)
.173 .802
Note: No sperm on microscopic TESE is the reference variable for logistic regression. Bernie. Micro-TESE in men with maturation arrest. Fertil Steril 2015.
4
findings seem to explain this higher SRR. We believe that the higher SRR in this group may occur because NOA in men with cryptorchidism results from damage to the testis, thereby producing large portions that have a complete loss of germ cells (i.e., Sertoli-cell-only histology and tubular atrophy). However, when present in these men, germ cells have the capacity for development to an advanced level of spermatogenesis; thus, these patients are likely to have other areas of the testis in which complete spermatogenesis occurs. Men with diffuse MA have worse SRR, compared with those who have focal MA in our series. This finding may not be surprising, given that men who have NOA have abnormal overall testicular function. Success of sperm retrieval depends on R1 area of the testis having better (complete) spermatogenesis. If the testis is uniformly abnormal in function, then mature sperm are unlikely to be present. Diffuse MA was additionally associated with larger testicular volume and higher FSH, compared with focal MA. Because of these findings, men who have diffuse MA can be particularly challenging to counsel preoperatively and treat. Although we do not recommend routine testicular biopsy for men who have NOA, this particular subset of men, i.e., those who have normal FSH, normal testicular volume, and azoospermia, may benefit from a biopsy as recommended by the American Urological Association Best Practice Statement (11). If these men are found to have diffuse or early MA, then they can be appropriately counseled on the low likelihood of success of their micro-TESE. If they have both FSH and testicular volume near the range of normal, these men may initially seem normal, until semen analysis shows azoospermia. Sperm retrieval rates may be overestimated in cases of normal FSH and/or testicular volume. Given the low SRR seen in men with normal FSH and normal testis volume, this subgroup clearly has the worst chance of sperm retrieval. Clinicians should be aware that men who have normal testicular volume and FSH, with NOA, may have particularly poor SRR outcomes, even with micro-TESE. Interestingly, we additionally identified a similar subgroup of men with Sertoli-cell-only syndrome, in which men with a normal testicular volume and an FSH of 10–15 mU/mL tended to have a lower SRR (12). Given the findings discussed, we sought to determine whether a further distinction could be drawn among men who have late MA, based on whether they have a focal or diffuse pattern. Men who had focal, early MA had better SRR outcomes than those who had diffuse, early MA (48% vs. 19%). However, this trend did not hold true for men who had late MA, as no difference in SRR was found among men with focal, late MA vs. diffuse, late MA (76% vs. 79%). These findings suggest that the positive prognostic indicator of late MA overwhelms the negative predictor of diffuse MA in these men. Late MA is inherently associated with a high chance of at least focal sperm production. Evidence for this association comes from the fact that late MA was predictive of successful retrieval on multivariate analysis, whereas focal MA was not. Although this difference could be due to the overall low numbers of diffuse MA cases, another possibility is that the effect of late MA development in the testis suggests VOL. - NO. - / - 2015
Fertility and Sterility® a higher likelihood of full spermatogenesis elsewhere in the tissue, compared with focal MA. Our multivariate analysis additionally revealed that elevated FSH was predictive of successful sperm retrieval. We have previously shown that elevated FSH can predict higher sperm retrieval, as it likely reflects heterogeneity of testicular histologies, which is critical to sperm retrieval in NOA (13). Another possibility is that men with higher FSH have smaller testes, increasing the chance of finding the specific tubules that contain sperm. Our study has both strengths and limitations. To our knowledge, this series of men who have MA is the largest that has been evaluated. However, the series is limited by its retrospective nature. Another limitation is the definition of diffuse vs. focal MA that was created for this subset of patients. Given that criteria to define diffuse vs. focal MA are not well established, we classified men as having diffuse MA only when all tubules on the intraoperative testis biopsy showed MA. Finally, given that we do not perform biopsies on men with NOA, the pathology used in this study is from intraoperative samples taken during micro-TESE. Although the information would be most useful preoperatively, a biopsy is clearly not indicated in most men who have NOA and are being evaluated for micro-TESE. The findings in this study can be used to preoperatively counsel patients for whom pathology data are available (either from a biopsy or previous TESE attempt), and have the known clinical characteristics (particularly in men with NOA, normal testis volume, and normal FSH). In conclusion, MA on diagnostic biopsy in men who have NOA is associated with an overall SRR of 52%, using microTESE. Men with late and focal MA have a higher SRR than do men with early and diffuse MA. Sperm retrieval in men with NOA depends on having heterogeneity in the testicular tissue. In the presence of MA, a high level of FSH may predict such heterogeneity. Among men who have MA, late MA may predict R1 region with complete sperm production and seems
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to be the best predictor of successful sperm retrieval with micro-TESE, compared with focal MA.
REFERENCES 1.
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Su LM, Palermo GD, Goldstein M, Veeck LL, Rosenwaks Z, Schlegel PN. Testicular sperm extraction with intracytoplasmic sperm injection for nonobstructive azoospermia: Testicular histology can predict success of sperm retrieval. J Urol 1999;161:112–6. Ezeh UI, Moore HD, Cooke ID. A prospective study of multiple needle biopsies versus a single open biopsy for testicular sperm extraction in men with non-obstructive azoospermia. Hum Reprod 1998;13:3075–80. Abdel Raheem A, Garaffa G, Rushwan N, De Luca F, Zacharakis E, Abdel Raheem T, et al. Testicular histopathology as a predictor of a positive sperm retrieval in men with non-obstructive azoospermia. BJU Int 2013; 111:492–9. 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. Ishikawa T, Fujioka H, Fujisawa M. Clinical and hormonal findings in testicular maturation arrest. BJU Int 2004;94:1314–6. Tsai MC, Cheng YS, Lin TY, Yang WH, Lin YM. Clinical characteristics and reproductive outcomes in infertile men with testicular early and late maturation arrest. Urology 2012;80:826–32. Weedin JW, Bennett RC, Fenig DM, Lamb DJ, Lipshultz LI. Early versus late maturation arrest: reproductive outcomes of testicular failure. J Urol 2011; 186:621–6. Schlegel PN. Testicular sperm extraction: Microdissection improves sperm yield with minimal tissue excision. Hum Reprod 1999;14:131–5. Ramasamy R, Schlegel PN. Microdissection testicular sperm extraction: effect of prior biopsy on success of sperm retrieval. J Urol 2007;177:1447–9. Hung AJ, King P, Schlegel PN. Uniform testicular maturation arrest: a unique subset of men with nonobstructive azoospermia. J Urol 2007;178:608–12. discussion 12. American Urological Association Education and Research, Inc. The evaluation of the azoospermic male: AUA best practice statement. Linthicum, MD: AUA; 2011. Berookhim BM, Palermo GD, Zaninovic N, Rosenwaks Z, Schlegel PN. Microdissection testicular sperm extraction in men with Sertoli cell-only testicular histology. Fertil Steril 2014;102:1282–6. Ramasamy R, Lin K, Gosden LV, Rosenwaks Z, Palermo GD, Schlegel PN. High serum FSH levels in men with nonobstructive azoospermia does not affect success of microdissection testicular sperm extraction. Fertil Steril 2009;92:590–3.
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ORIGINAL ARTICLE: ANDROLOGY
SUPPLEMENTAL TABLE 1 Univariate analysis of factors associated with positive sperm retrieval. Factor
Exp(B)
95% CI
P value
Age (y) Testicular volume (cc) Testosterone Estradiol LH FSH Early (reference) vs. late maturation arrest Diffuse (reference) vs. focal Orchiopexy Klinefelter
1.033 0.974 1.339 1.003 1.563 2.936 0.181
(0.993, 1.074) (0.924, 1.026) (0.324, 5.534) (0.982, 1.023) (0.395, 6.191) (1.331, 6.477) (0.092, 0.356)
.103 .319 .687 .811 .525 .008 < .001
2.477
(1.269, 4.833)
.008
2.078 >1,000
(1.070, 4.037) (0.000, unable to calculate)
.031 .999
Note: No sperm on microscopic TESE is the reference variable for logistic regression. Bernie. Micro-TESE in men with maturation arrest. Fertil Steril 2015.
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Outcomes of microdissection testicular sperm extraction in men with nonobstructive azoospermia due to maturation arrest Aaron M. Bernie, M.D., M.P.H.,a Kalee Shah, B.A.,a Joshua A. Halpern, M.D.,a Jason Scovell, B.A.,b Ranjith Ramasamy, M.D.,b Brian Robinson, M.D.,c and Peter N. Schlegel, M.D.a a Department of Urology, Weill Cornell Medical College, New York, New York; b Baylor College of Medicine, Houston, Texas; and c Department of Pathology & Laboratory Medicine, Weill Cornell Medical College, New York, New York
Objective: To evaluate sperm retrieval in men with nonobstructive azoospermia and maturation arrest (MA) undergoing microdissection testicular sperm extraction (micro-TESE). Design: Retrospective chart review. Setting: Tertiary referral center. Patient(s): Men with nonobstructive azoospermia and MA who underwent micro-TESE. Intervention(s): Microdissection TESE. Main Outcome Measure(s): Sperm retrieval rate (SRR). Result(s): A total of 211 patients (13%) had a histologic finding of MA at the most advanced level. The overall SRR was 52%. A total of 146 patients were classified as having early MA (arrest at the primary spermatocyte stage), and 65 as having late MA (early spermatid stage). The SRR in men with early, vs. late, MA was 40% vs. 78%. Of the 211 men with MA, 51 had diffuse MA (100% of tubules showed MA). The SRR was significantly lower in men with diffuse vs. focal MA (35% vs. 57%). On multivariable analysis, late MA and higher follicle-stimulating hormone levels were positively associated with successful sperm retrieval. Conclusion(s): Sperm were successfully identified in up to one half of the men with MA after micro-TESE. Among men with MA, late MA seems to be the best predictor of successful sperm retrieval with micro-TESE. (Fertil SterilÒ Use your smartphone 2015;-:-–-. Ó2015 by American Society for Reproductive Medicine.) to scan this QR code Key Words: Microdissection testicular sperm extraction, maturation arrest, nonobstructive and connect to the azoospermia Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/berniea-micro-tese-maturation-arrest/
INTRODUCTION Nonobstructive azoospermia (NOA), or testicular failure, affects 1% of males and 10% of those who seek fertility assistance (1). Three histopathological patterns are typically described in the biopsies of men who have NOA: reduced spermatogenesis with all germ cell types present (hypospermato-
genesis); premature arrest of spermatogenesis (maturation arrest [MA]); and complete absence of germ cells (Sertoli-cell-only syndrome) (2). Limited biopsies may fail to identify focal areas of sperm production that may otherwise be found with a detailed micro– testicular sperm extraction (TESE) search. Clinically, the histologic classi-
Received April 7, 2015; revised May 13, 2015; accepted May 29, 2015. A.M.B. has nothing to disclose. K.S. has nothing to disclose. J.A.H. has nothing to disclose. J.S. has nothing to disclose. R.R. has nothing to disclose. B.R. has nothing to disclose. P.N.S. has nothing to disclose. Reprint requests: Peter N. Schlegel, M.D., Department of Urology, Brady Urologic Health Center, 525 East 68th St, Starr Pavilion, 9th Floor, New York, New York 10065 (E-mail: [email protected]. edu). Fertility and Sterility® Vol. -, No. -, - 2015 0015-0282/$36.00 Copyright ©2015 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2015.05.037 VOL. - NO. - / - 2015
discussion forum for this article now.*
* Download a free QR code scanner by searching for “QR scanner” in your smartphone’s app store or app marketplace.
fication on a diagnostic biopsy can provide value in predicting the chance of success with TESE. For instance, the average sperm retrieval rate (SRR) is 94% in patients with hypospermatogenesis, compared with 40% in patients with Sertoli-cell-only syndrome, even with micro-TESE (3, 4). Maturation arrest is defined as an absence of mature spermatozoa as a result of an early arrest in germ cell development in the seminiferous tubules. It is further delineated into early and late MA, based on the mostmature cell type noted on histology. In early MA, spermatogenic arrest occurs at the spermatogonia or spermatocyte stage; in late MA, arrest occurs at the 1
ORIGINAL ARTICLE: ANDROLOGY spermatid stage (5–7). Late MA is distinguished from hypospermatogenesis in that no condensed, oval spermatids are seen in late MA. The underlying genetic causes that result in the phenotypes of early vs. late MA remain undetermined. On biopsy, MA can also vary in its heterogeneity, from focal to diffuse, further complicating prediction of SRR with TESE. In men with MA, studies show a highly variable SRR of 23%–50% (6, 7). Both Tsai et al. (6) and Weedin et al. (7) found that, compared with men with late MA, those diagnosed with early MA had a decreased probability of successful sperm retrieval. Due to the limited number of cases in prior publications, histopathology could not be used to predict successful TESE. Therefore, we present a series of patients with NOA who underwent micro-TESE, performed by a single surgeon at our center, and were diagnosed with MA. We classified men with MA as either early or late, and focal or diffuse, and evaluated sperm SRR.
METHODS After receiving approval from the Weill Cornell Medical College Institutional Review Board, we retrospectively reviewed the charts of 1,686 consecutive patients who had NOA, confirmed by analysis of 2 centrifuged semen samples, from November 1995 through June 2014. All patients who were reviewed underwent micro-TESE at a single center, performed by a single surgeon. The NOA was confirmed via repeat semen analysis on the day of micro-TESE. Men with complete azoospermia factor (AZF)a or AZFb microdeletions were excluded from our analysis: no men with AZFa or AZFb underwent micro-TESE. Preoperatively identifiable factors, including age, follicle-stimulating hormone (FSH), total serum testosterone (T), testis volume on physical exam, presence of a varicocele, history of cryptorchidism, history of testicular cancer, prior diagnosis of Klinefelter syndrome, and Y microdeletion (AZFc) were analyzed. Men with a history of varicocele underwent varicocelectomy before micro-TESE. Micro-TESE
was performed as described previously (8). All men underwent initial unilateral micro-TESE, and if no sperm were retrieved, the contralateral testis was explored. Sperm retrieved were cryopreserved according to patient preference. Although some patients were referred to our center with a previous diagnostic biopsy or TESE, we did not perform routine biopsies because a diagnostic biopsy does not predict the presence of focal areas of spermatogenesis that microTESE can identify (9). Given the limited information available from previous outside biopsies, and the small number of men who had them performed, histologic data from outside biopsies were not included in this analysis. The pathologic information used in this study was from a single random intraoperative biopsy taken at the time of micro-TESE. Patients were considered to have MA if the most advanced pathologic pattern on either diagnostic or random intraoperative testis biopsy was consistent with MA. Biopsy slides were then re-reviewed by our uropathologist, who was blinded to the outcome of micro-TESE, and classified as either early or late MA. Early MA was defined as arrest of spermatogenesis at the primary spermatocyte stage (nuclei with filamentous chromatin, Fig. 1). Late MA was defined as arrest of spermatogenesis at the early spermatid phase (dark round nuclei, Fig. 1) and the absence of elongated spermatids/spermatozoa (elongated/oval nuclei). Although previous studies have looked at men who have MA (6, 7, 10), no well-defined criteria have been established for determining if a patient is considered to have diffuse or focal MA. Thus, we established a classification scheme intended to delineate the 2 groups. Given that all pathology was from a single intraoperative biopsy taken during microTESE, we felt that if a biopsy showed 100% MA, i.e., all tubules examined had MA, then those patients likely had only MA in the remainder of the testis. In contrast, if a portion of the biopsy revealed an alternate pathology (i.e., Sertolicell-only syndrome or hypospermatogenesis), we classified those men as having focal MA. Successful sperm retrieval depends on having an area of the testis where complete
FIGURE 1
Early (left) and late maturation arrest (right). Bernie. Micro-TESE in men with maturation arrest. Fertil Steril 2015.
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Fertility and Sterility® univariate analysis were included for multivariable binary logistic regression. All statistical analysis was performed using SPSS, version 22 (IBM).
TABLE 1 Early vs. late maturation arrest. Variable n Sperm retrieval rate Male age (y) Female age (y) Average testicular volume (cc) FSH (mU/mL) Testosterone (ng/dL) Y microdeletion Cryptorchidism Klinefelter Varicocele Cancer Hx
All MA patients
Early MA
Late MA
P value
211 52 36 32 10
146 40 36 32 10
65 78 37 32 10
< .0001 .53 .96 .84
21 408 12 23 0 68 3
22 400 13 18 1 66 5
20 426 9 34 0 71 0
.22 .33 .58 .024 .86 .64 .17
RESULTS Overall Characteristics
Note: Values are %, or median, unless otherwise indicated. Bernie. Micro-TESE in men with maturation arrest. Fertil Steril 2015.
spermatogenesis occurs. If the testis is uniformly abnormal in function, such as that seen with a testis that is 100% MA, then mature sperm are unlikely to be present. Therefore, in this study, patients were classified as having diffuse MA if 100% of the tubules revealed MA, and as having focal MA if <100% of the tubules revealed MA. We performed both univariate and multivariable binary logistic regression analysis to identify factors associated with and predictive of positive sperm retrieval during a micro-TESE. For univariate analysis, we included subject age, testicular volume, serum T, luteinizing hormone (LH), FSH, early vs. late MA, diffuse vs. focal MA, history of cryptorchidism, and history of Klinefelter syndrome. We tested for data normality using a quantile-quantile (Q-Q) plot, along with the Shapiro-Wilk test. Non-normally distributed continuous variables were normalized using a logistic transformation and include T, FSH, and LH. Factors that were significantly (P< .05) associated with sperm retrieval on the
A total of 211 patients (13% of the total micro-TESE cohort) were found to have a most advanced histologic finding of MA on pathologic analysis. The median age was 36 (7) years. Median testicular volume was 10 (5) cc. The overall SRR for the cohort was 52%, with a clinical pregnancy rate of 44%. Characteristics for all MA patients are included in Table 1.
Early vs. Late Maturation Arrest Of the 211 men, 146 patients (69%) were classified as having early MA, and 65 (31%) were classified as having late MA, on pathology review (Fig. 2). Table 1 presents the characteristic differences between early and late MA. The SRR was higher in men who had late, vs. early, MA (78% vs. 40%; P< .0001). Men who had late MA were more likely to have a history of cryptorchidism, compared with men who had early MA (34% vs. 23%, P¼ .024). No differences were found in male or female age, testicular volume, FSH or T, diagnosis of Y microdeletion or Klinefelter's syndrome, varicocele, or history of cancer in men with early vs. late MA.
Diffuse vs. Focal Maturation Arrest Of the 211 men who had MA, 51 (24%) had diffuse MA (Fig. 2). The SRR was significantly lower in men with diffuse MA, compared with men with focal MA (35% vs. 57%, P¼ .008). Men with diffuse MA additionally had larger testicular size (13 vs. 8.7 cc, P< .001) and lower FSH levels (14 vs. 25 mIU/mL, P< .001). Men with focal MA were more likely to have a history of cryptorchidism, compared with men with diffuse MA (30% vs. 10%, P¼ .004).
FIGURE 2
Breakdown of men who had NOA and MA, into early vs. late, and diffuse vs. focal, MA. MA ¼ maturation arrest; NOA ¼ nonobstructive azoospermia; SRR ¼ sperm retrieval rate. Bernie. Micro-TESE in men with maturation arrest. Fertil Steril 2015.
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ORIGINAL ARTICLE: ANDROLOGY Predictors of Successful Sperm Retrieval On univariate linear regression analysis, we found that higher FSH, late MA, focal MA, and a history of cryptorchidism were positively associated with successful sperm retrieval (P< .05) (Supplemental Table 1, available online). However, on multivariable analysis, only late MA and higher FSH were positively associated with successful sperm retrieval (P< .05) (Table 2).
DISCUSSION Men who have NOA and MA represent a challenging group of patients to treat. In particular, the decision to pursue microTESE, and the determination of potential for SRR, can be complex. Thus, identification of clinical characteristics that can help predict micro-TESE outcome is valuable for clinical management and preoperative counseling of these patients, particularly those who need a repeat procedure after previous failed sperm retrieval. Although we do not advocate routine preoperative biopsies for men with NOA, if a previous biopsy or pathology from attempted TESE are available, they can be used in counseling. In such cases, we recommend that pathology from prior procedures be closely re-reviewed by a dedicated genitourinary pathologist to identify focal/diffuse and early/late MA, as these are critical to predicting the success of subsequent micro-TESE. We evaluated sperm retrieval in men who had MA and found an overall SRR of 52%. Men who had late and focal MA were more likely to have successful sperm retrieval than were men who had early and diffuse MA, respectively. On a multivariate analysis, both elevated FSH and late MA were found to be predictive of successful sperm retrieval. We found a higher SRR rate in men who had late MA, compared with men who had early MA (6, 7). Men who had late MA in our series were almost twice as likely to have sperm retrieved, compared with men who had early MA (78% vs. 40%). Late MA on diagnostic biopsy clearly suggests that the more advanced the pattern seen on biopsy, the more likely there are to be areas of complete spermatogenesis elsewhere in the testicle. Interestingly, men with a history of cryptorchidism were more likely to have late MA vs. early MA in our series, and the subset of men with both cryptorchidism and late MA had the highest SRR of any subgroup. No previous
TABLE 2 Multivariable factors predictive of finding sperm on microscopic TESE. Factor FSH Early (reference) vs. late maturation arrest Diffuse (reference) vs. focal Orchiopexy
Odds ratio
95% CI
P value
2.978 0.169
(1.061, 8.356) (0.080, 0.357)
.038 < .001
1.732 1.106
(0.786, 3.818) (0.504, 2.428)
.173 .802
Note: No sperm on microscopic TESE is the reference variable for logistic regression. Bernie. Micro-TESE in men with maturation arrest. Fertil Steril 2015.
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findings seem to explain this higher SRR. We believe that the higher SRR in this group may occur because NOA in men with cryptorchidism results from damage to the testis, thereby producing large portions that have a complete loss of germ cells (i.e., Sertoli-cell-only histology and tubular atrophy). However, when present in these men, germ cells have the capacity for development to an advanced level of spermatogenesis; thus, these patients are likely to have other areas of the testis in which complete spermatogenesis occurs. Men with diffuse MA have worse SRR, compared with those who have focal MA in our series. This finding may not be surprising, given that men who have NOA have abnormal overall testicular function. Success of sperm retrieval depends on R1 area of the testis having better (complete) spermatogenesis. If the testis is uniformly abnormal in function, then mature sperm are unlikely to be present. Diffuse MA was additionally associated with larger testicular volume and higher FSH, compared with focal MA. Because of these findings, men who have diffuse MA can be particularly challenging to counsel preoperatively and treat. Although we do not recommend routine testicular biopsy for men who have NOA, this particular subset of men, i.e., those who have normal FSH, normal testicular volume, and azoospermia, may benefit from a biopsy as recommended by the American Urological Association Best Practice Statement (11). If these men are found to have diffuse or early MA, then they can be appropriately counseled on the low likelihood of success of their micro-TESE. If they have both FSH and testicular volume near the range of normal, these men may initially seem normal, until semen analysis shows azoospermia. Sperm retrieval rates may be overestimated in cases of normal FSH and/or testicular volume. Given the low SRR seen in men with normal FSH and normal testis volume, this subgroup clearly has the worst chance of sperm retrieval. Clinicians should be aware that men who have normal testicular volume and FSH, with NOA, may have particularly poor SRR outcomes, even with micro-TESE. Interestingly, we additionally identified a similar subgroup of men with Sertoli-cell-only syndrome, in which men with a normal testicular volume and an FSH of 10–15 mU/mL tended to have a lower SRR (12). Given the findings discussed, we sought to determine whether a further distinction could be drawn among men who have late MA, based on whether they have a focal or diffuse pattern. Men who had focal, early MA had better SRR outcomes than those who had diffuse, early MA (48% vs. 19%). However, this trend did not hold true for men who had late MA, as no difference in SRR was found among men with focal, late MA vs. diffuse, late MA (76% vs. 79%). These findings suggest that the positive prognostic indicator of late MA overwhelms the negative predictor of diffuse MA in these men. Late MA is inherently associated with a high chance of at least focal sperm production. Evidence for this association comes from the fact that late MA was predictive of successful retrieval on multivariate analysis, whereas focal MA was not. Although this difference could be due to the overall low numbers of diffuse MA cases, another possibility is that the effect of late MA development in the testis suggests VOL. - NO. - / - 2015
Fertility and Sterility® a higher likelihood of full spermatogenesis elsewhere in the tissue, compared with focal MA. Our multivariate analysis additionally revealed that elevated FSH was predictive of successful sperm retrieval. We have previously shown that elevated FSH can predict higher sperm retrieval, as it likely reflects heterogeneity of testicular histologies, which is critical to sperm retrieval in NOA (13). Another possibility is that men with higher FSH have smaller testes, increasing the chance of finding the specific tubules that contain sperm. Our study has both strengths and limitations. To our knowledge, this series of men who have MA is the largest that has been evaluated. However, the series is limited by its retrospective nature. Another limitation is the definition of diffuse vs. focal MA that was created for this subset of patients. Given that criteria to define diffuse vs. focal MA are not well established, we classified men as having diffuse MA only when all tubules on the intraoperative testis biopsy showed MA. Finally, given that we do not perform biopsies on men with NOA, the pathology used in this study is from intraoperative samples taken during micro-TESE. Although the information would be most useful preoperatively, a biopsy is clearly not indicated in most men who have NOA and are being evaluated for micro-TESE. The findings in this study can be used to preoperatively counsel patients for whom pathology data are available (either from a biopsy or previous TESE attempt), and have the known clinical characteristics (particularly in men with NOA, normal testis volume, and normal FSH). In conclusion, MA on diagnostic biopsy in men who have NOA is associated with an overall SRR of 52%, using microTESE. Men with late and focal MA have a higher SRR than do men with early and diffuse MA. Sperm retrieval in men with NOA depends on having heterogeneity in the testicular tissue. In the presence of MA, a high level of FSH may predict such heterogeneity. Among men who have MA, late MA may predict R1 region with complete sperm production and seems
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to be the best predictor of successful sperm retrieval with micro-TESE, compared with focal MA.
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Su LM, Palermo GD, Goldstein M, Veeck LL, Rosenwaks Z, Schlegel PN. Testicular sperm extraction with intracytoplasmic sperm injection for nonobstructive azoospermia: Testicular histology can predict success of sperm retrieval. J Urol 1999;161:112–6. Ezeh UI, Moore HD, Cooke ID. A prospective study of multiple needle biopsies versus a single open biopsy for testicular sperm extraction in men with non-obstructive azoospermia. Hum Reprod 1998;13:3075–80. Abdel Raheem A, Garaffa G, Rushwan N, De Luca F, Zacharakis E, Abdel Raheem T, et al. Testicular histopathology as a predictor of a positive sperm retrieval in men with non-obstructive azoospermia. BJU Int 2013; 111:492–9. 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. Ishikawa T, Fujioka H, Fujisawa M. Clinical and hormonal findings in testicular maturation arrest. BJU Int 2004;94:1314–6. Tsai MC, Cheng YS, Lin TY, Yang WH, Lin YM. Clinical characteristics and reproductive outcomes in infertile men with testicular early and late maturation arrest. Urology 2012;80:826–32. Weedin JW, Bennett RC, Fenig DM, Lamb DJ, Lipshultz LI. Early versus late maturation arrest: reproductive outcomes of testicular failure. J Urol 2011; 186:621–6. Schlegel PN. Testicular sperm extraction: Microdissection improves sperm yield with minimal tissue excision. Hum Reprod 1999;14:131–5. Ramasamy R, Schlegel PN. Microdissection testicular sperm extraction: effect of prior biopsy on success of sperm retrieval. J Urol 2007;177:1447–9. Hung AJ, King P, Schlegel PN. Uniform testicular maturation arrest: a unique subset of men with nonobstructive azoospermia. J Urol 2007;178:608–12. discussion 12. American Urological Association Education and Research, Inc. The evaluation of the azoospermic male: AUA best practice statement. Linthicum, MD: AUA; 2011. Berookhim BM, Palermo GD, Zaninovic N, Rosenwaks Z, Schlegel PN. Microdissection testicular sperm extraction in men with Sertoli cell-only testicular histology. Fertil Steril 2014;102:1282–6. Ramasamy R, Lin K, Gosden LV, Rosenwaks Z, Palermo GD, Schlegel PN. High serum FSH levels in men with nonobstructive azoospermia does not affect success of microdissection testicular sperm extraction. Fertil Steril 2009;92:590–3.
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ORIGINAL ARTICLE: ANDROLOGY
SUPPLEMENTAL TABLE 1 Univariate analysis of factors associated with positive sperm retrieval. Factor
Exp(B)
95% CI
P value
Age (y) Testicular volume (cc) Testosterone Estradiol LH FSH Early (reference) vs. late maturation arrest Diffuse (reference) vs. focal Orchiopexy Klinefelter
1.033 0.974 1.339 1.003 1.563 2.936 0.181
(0.993, 1.074) (0.924, 1.026) (0.324, 5.534) (0.982, 1.023) (0.395, 6.191) (1.331, 6.477) (0.092, 0.356)
.103 .319 .687 .811 .525 .008 < .001
2.477
(1.269, 4.833)
.008
2.078 >1,000
(1.070, 4.037) (0.000, unable to calculate)
.031 .999
Note: No sperm on microscopic TESE is the reference variable for logistic regression. Bernie. Micro-TESE in men with maturation arrest. Fertil Steril 2015.
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