Testicular Mapping Biopsy in Non-obstructive Azoospermia (NOA) Patients: A Case Series from Indonesia

The value of repeat testicular sperm retrieval in azoospermic men

What is the added value of enzymatic processing of testicular biopsies on testicular sperm retrieval (SR) rates for patients with non-obstructive azoospermia (NOA)? In addition to mechanical mincing, enzymatic digestion increased SR rates in testicular biopsies of NOA patients. Many studies focus on the surgical approach to optimize recovery of testicular sperm in NOA, and in spite of that, controversy still exists about whether the type of surgery makes any difference as long as multiple biopsies are taken. Few studies, however, focus on the role of the IVF laboratory and the benefit of additional lab procedures, e.g. enzymatic digestion, in order to optimize SR rates. This retrospective single-center cohort study included all patients who underwent their first testicular sperm extraction (TESE) by open multiple-biopsy method between January 2004 and July 2022. Only patients with a normal karyotype, absence of Y-q deletions and a diagnosis of NOA based on histology were included. The primary outcome was SR rate after mincing and/or enzymes. The secondary outcome was cumulative live birth (CLB) after ICSI with fresh TESE and subsequent ICSI cycles with frozen TESE. Multiple biopsies were obtained from the testis, unilaterally or bilaterally, on the day of oocyte retrieval. Upon mechanical mincing, biopsies were investigated for 30 min; when no or insufficient numbers of spermatozoa were observed, enzymatic treatment was performed using Collagenase type IV. Multivariable regression analysis was performed to predict CLB per TESE by adjusting for the following confounding factors: male FSH level, female age, and requirement of enzymatic digestion to find sperm. We included 118 patients, of whom 72 (61.0%) had successful SR eventually. Spermatozoa were retrieved after mechanical mincing for 28 patients (23.7%; 28/118) or after additional enzymatic digestion for another 44 patients (37.2%; 44/118). Thus, of the 90 patients requiring enzymatic digestion, sperm were retrieved for 44 (48.9%). Male characteristics were not different between patients with SR after mincing or enzymatic digestion, in regard to mean age (34.5 vs 34.5 years), testis volume (10.2 vs 10.6 ml), FSH (17.8 vs 16.9 IU/l), cryptorchidism (21.4 vs 34.1%), varicocele (3.6 vs 4.6%), or histological diagnosis (Sertoli-cell only 53.6 vs 47.7%, maturation arrest 21.4 vs 38.6%, sclerosis/atrophy 25.0 vs 13.6%).Of the 72 patients with sperm available for ICSI, 23/72 (31.9%) achieved a live birth (LB) after the injection with fresh testicular sperm (and fresh or frozen embryo transfers). Of the remaining 49 patients without LB, 34 (69.4%) had supernumerary testicular sperm frozen. Of these 34 patients, 19 (55.9%) continued ICSI with frozen testicular sperm, and 9/19 (47.4%) had achieved an LB after ICSI with frozen testicular sperm. Thus, the total CLB was 32/118 (27.1%) per TESE or 32/72 (44.4%) per TESE with sperm retrieved.Of the female characteristics (couples with sperm available), only female age (30.3 vs 32.7 years; P = 0.042) was significantly lower in the group with a LB, compared to those without.The CLB with testicular sperm obtained after enzymatic digestion was 31.8% (14/44), while the CLB with sperm obtained after mincing alone was 64.3% (18/28). Multivariable logistic regression analysis showed that when enzymatic digestion was required, it was associated with a significant decrease in CLB per TESE (OR: 0.23 (0.08-0.7); P = 0.01). Limitations of the study are related to the retrospective design. However, the selection of only patients with NOA, and specific characteristics (normal karyotype and absence Y-q deletion) and having their first TESE, strengthens our findings. Enzymatic processing increases the SR rate from testicular biopsies of NOA patients compared to mechanical mincing only, demonstrating the importance of an appropriate laboratory protocol. However, NOA patients should be counseled that when sperm have been found after enzymatic digestion, their chances to father a genetically own child may be lower compared to those not requiring enzymatic digestion. None reported. N/A.

Study question How does applying the APHRODITE criteria impact the sperm retrieval rate in non-obstructive azoospermia (NOA) patients undergoing micro-TESE? Summary answer The APHRODITE could classify poor prognostic patients and predict the sperm retrieval rates in micro-TESE, highlighting its role in patient selection and counseling. What is known already Microsurgical testicular sperm extraction (micro-TESE) is the gold standard for sperm retrieval in non-obstructive azoospermia (NOA), but predicting sperm retrieval success remains challenging. The novel APHRODITE is a simple criterion for patients with male infertility based on clinical and hormonal factors to improve the reproductive outcome. However, data on applying these criteria to NOA populations remains limited. This study evaluated the role of the APHRODITE criteria in micro-TESE outcomes. Study design, size, duration The study was designed as a cross-sectional study. We retrospectively analyzed the outcome of 205 NOA patients who underwent Micro-TESE at IVFTA HCM, Tam Anh General Hospital, Vietnam, from 2021 to 2023. Participants/materials, setting, methods All patients underwent comprehensive clinical and laboratory evaluation for non-obstructive azoospermia (NOA), including hormonal profiling, genetic testing, and testicular volume assessment. Patients were classified according to the APHRODITE criteria into Groups 2, 3, and 4, based on clinical and biochemical prognostic factors. Microsurgical testicular sperm extraction (micro-TESE) was performed for all patients. Multivariate logistic regression analysis evaluated the association between patient variables, including age, clinical factors, genetic markers, APHRODITE group, and sperm retrieval success. Main results and the role of chance We found no significant differences in clinical factors or hormonal profiles associated with sperm retrieval success, except in patients with Klinefelter syndrome, who demonstrated a significantly lower sperm retrieval rate (SRR = 6% [2/17], p = 0.026). The overall SRR among non-obstructive azoospermia (NOA) patients was 40%. Using the APHRODITE criteria, Group 4 included the most significant proportion of patients (77.9%, 158/205) but demonstrated the lowest SRR of 36%, compared with Group 2 (SRR = 62.9%) and Group 3 (SRR = 40%, p = 0.031). Interestingly, while Follicle-stimulating hormone (FSH) and Testosterone concentrations were not significantly associated with SRR, multivariate logistic regression analysis revealed significant findings. APHRODITE group 4 was associated with significantly lower sperm retrieval (OR: 4.43, 95% CI: 1.62–12.16, p = 0.0038). Group 3 showed decreased retrieval rates, although not statistically significant (OR: 3.02, 95% CI: 0.87–10.53, p = 0.082). Increasing age was associated with retrieval failure (OR: 0.95, 95% CI: 0.91–0.99, p = 0.032). Other variables, such as testicular volume, genetic markers, history of mumps, varicocele surgery, Klinefelter syndrome and previous unsuccessful retrieval attempts, were not significant predictors. Limitations, reasons for caution The study limitations include the retrospective collection, which can lead to selection bias. Additionally, the small sample size and the applicability of the results to all NOAs should be considered. Wider implications of the findings These findings highlight the usefulness of the APHRODITE criteria among NOA patient underwent micro-TESE, especially for Group 4 patients, which may enhance patient counseling, optimize surgical planning, and refine treatment strategies for NOA, contributing to more effective and personalized fertility care. Trial registration number No

Study question What is the added value of enzymatic processing of testicular biopsies on testicular sperm retrieval rates in patients with non-obstructive azoospermia (NOA)? Summary answer In addition to mechanical mincing, enzymatic digestion increased sperm retrieval rates in testicular biopsies of NOA patients with 48.9%. What is known already Many studies focus on the surgical approach to optimize recovery of testicular sperm in NOA, and in spite of that, there is still controversy whether the type of surgery makes any difference as long as multiple biopsies are taken. Few studies, however, focus on the role of the IVF laboratory and the benefit of additional lab procedures, like enzymatic digestion, in order to optimize sperm retrieval rates and CLB rate per TESE. Study design, size, duration A retrospective single-center cohort study including all patients who underwent their first TESE by open multiple-biopsy method from January 2004 till July 2022. Only patients with a normal karyotype, absence of Y-q deletions and a strict diagnosis of NOA based on histology were included. Primary outcome was sperm retrieval after mincing or enzymes for intracytoplasmic sperm injection (ICSI). Secondary outcome was CLB after ICSI with fresh TESE, and subsequent ICSI cycles with frozen TESE. Participants/materials, setting, methods Multiple biopsies were obtained from the testis, unilateral or bilateral, on the day of oocyte retrieval. Upon mechanical mincing, dishes were searched for 30 min; when no or insufficient numbers of spermatozoa were observed, enzymatic treatment was performed using collagenase type IV. Multivariable regression analysis was performed to predict CLB by adjusting for the following confounding factors: male age, male FSH level, cryptorchidism, enzymatic digestion, number of oocytes and female age. Main results and the role of chance Hundred-eighteen patients were included of which 61.0% had successful sperm retrieval. Spermatozoa were retrieved after mechanical mincing (23.7%; 28/118) or after additional enzymatic digestion of the remaining 90 patients (48.9%; 44/90). Mean male characteristics were not different between patients with sperm retrieval after mincing or enzymes: age (34.5 vs 34.5 y), testis volume (10.2 vs 10.6 ml), FSH (17.8 vs 16.9 IU/l), cryptorchidism (21.4 vs 34.1%) and histological diagnosis (Sertoli Cell Only 53.6 vs 47.7%, maturation arrest 21.4 vs 38.6%, sclerosis/atrophy 25.0 vs 13.6%), respectively. Of the 72 patients with sperm available for ICSI, 23/72 (31.9%) obtained a LB after the injection with fresh testicular sperm (fresh and frozen embryo transfers). Forty-nine patients remained without LB, of which 34 had supernumerary testicular sperm frozen. Of these, 9/47 (47.4%) had a LB after ICSI with frozen testicular sperm, giving rise to a total CLB per TESE of 32/118 (27.1%) or 32/72 (44.4%) CLB per TESE with sperm. Of the female characteristics (couples with sperm available), only female age (30.3 vs 32.7 y – p = 0,042) was significantly lower in the group with a live birth. Multivariable logistic regression analysis showed that enzymatic digestion was associated with significant decrease of CLB per TESE. Limitations, reasons for caution Limitations of the study are related to the retrospective design. The selection of only NOA patients with specific characteristics (normal karyotype and absence Y-q deletion) and having their first TESE ever strengthens our findings. Whether enzymatic digestion after a failed TESE without digestion may improve retrieval rate remains undecided. Wider implications of the findings Enzymatic processing increases the sperm retrieval rate from testicular biopsies of NOA patients compared to mechanical mincing, demonstrating the importance of an appropriate laboratory protocol. NOA patients should be counselled that if sperm has been found after enzymatic digestion, their chances to father a genetically own child will be lower. Trial registration number not applicable

Early Versus Late Maturation Arrest: Reproductive Outcomes of Testicular Failure

Spermatozoa can be retrieved in non-obstructive azoospermia (NOA) patients despite the absence of ejaculated spermatozoa in their semen because of the presence of isolated foci with active spermatogenesis. Conventional testicular sperm extraction (c-TESE) in patients with NOA has been partially replaced by micro-TESE. It is still under debate the problem regarding the higher costs related to micro-TESE when compared with c-TESE. In this study, we evaluated sperm retrieval rate (SRR) of c-TESE in naive NOA patients. Sixty-three NOA patients were referred to our centre for a c-TESE. For every subject, we collected demographic data, cause of infertility, time to first infertility diagnosis, serum levels of LH, FSH, total testosterone and prolactin. A statistical analysis was conducted to correlate all the clinical variables, the histology and the Johnsen score with the SRR. Sixty-three consecutive NOA patients with a mean age of 37.3years were included. The positive SRR was 47.6%. No statistical differences were observed between positive vs. negative SRR regarding mean FSH (17.12 vs. 19.03 mUI/mL; p=0.72), and LH (9.72 vs. 6.92 mUI/mL; p=0.39) values. Interestingly, we found a statistically significant difference in terms of time to first infertility diagnosis (+SRR vs. -SRR; 44.5 vs. 57months; p=0.02) and regarding to age (+SSR vs. -SRR; 40.1 vs. 35.3; p=0.04). There was a statistically significant decrease in SRRs with the decline in testicular histopathology from hypospermatogenesis to maturation arrest, and SCO. The mean Johnsen score was 5.9 with a mean percentage of Johnsen score ≥8 tubules equal to 19%. The overall pregnancy rate was 26.6%. In our prospective cohort of patients successful SRR with c-TESE was 47.6%. Lower costs and high reproducibility of this technique still support this procedure as an actual reliable option in NOA patients for sperm retrieval.

BackgroundMicrodissection testicular sperm extraction (micro-TESE) is an effective method to retrieve sperm from non-obstructive azoospermia (NOA) patients. However, the predictive factors for sperm retrieval rate (SRR) remain confused. The goal of our study was to identify the role of testicular pathological morphometric parameters, including diameter of tubule (DT), height of spermatogenic epithelium (HSE), and thickness of basement-membrane (TBM) in NOA patients, and to develop a predictive model and nomogram to predict SRR based on these morphometric parameters.MethodsThis study involved two cohorts including 406 men with NOA. A retrospective cohort of 313 males with NOA who underwent micro-TESE at Northwest Women’s and Children’s Hospital (Xi’an, China) were included to build a prediction model of SRR. Then, another retrospective cohort of 93 males with NOA from Ren Ji Hospital (Shanghai, China) were recruited to validate the prediction model. The measurement of testicular morphometric parameters as well as the assessment of Johnsen score and pathological diagnostic types were performed by at least two pathologists. Testicular volumes as well as level of serum hormones including follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) were also measured. Logistic regressions were used to test potential predictors of SRR. Area under curve (AUC) estimates was used to evaluate the predictive accuracy. The validation datasets were used to validate the prediction model by prediction accuracy.ResultsOur study demonstrated that DT and HSE were significantly longer in successful sperm retrieval group than in failed sperm retrieval group. In addition, DT and HSE were positively correlated with Johnsen score, testicular volume, and serum T, while, were negatively correlated with serum FSH and serum LH. On the contrary, TBM demonstrated exact opposite results. Moreover, univariate logistic analyses illustrated that longer DT and HSE was associated with a high SRR, respectively. Further multivariate logistic analyses constructed multi-variables models with better predictive abilities compared with single-variables models. A multi-variables model (predicting score = -0.612–0.018 × DT + 0.040 × HSE + 0.097 × Johnsen score-0.004 × serum FSH) was finally constructed with the best predictive ability (AUC = 0.839, sensitivity = 71.4% specificity = 77.5%, cut-off value = 0.489). A higher predicting score indicated a higher possibility of successful sperm retrieval. The predictive accuracy was 89.25% in the external validation dataset.ConclusionWe report for the first time that DT and HSE have pretty ability to predict SRR in NOA patients.

Low levels of serum inhibin B do not exclude successful sperm recovery in men with nonmosaic Klinefelter syndrome

The treatment of male factor infertility, due to non-obstructive azoospermia (NOA), by testicular sperm extraction (TESE)–ICSI is a major breakthrough in assisted reproduction. However, although the first patient series on this approach were published more than 15 years ago (Tournaye et al., 1995), to date, it still remains difficult to provide candidate patients with truthful data about their chances to eventually father their genetically own child. In this issue, Boitrelle et al. (2011) published a study trying to determine predictive parameters to improve patient counselling. Retrieval rates after testicular surgery reported in the literature differ considerably. While in unselected NOA patients, the surgical approach seems to have a limited effect as long as multiple biopsies are taken whenever needed, the extended processing of the wet preparations of the testicular tissue may have a greater impact on sperm recovery. Unfortunately, large well-designed controlled trials to support both assertions are lacking. But sperm retrieval rates are also subject to the (pre)selection of patients and can be biased either by re-allocating successful patients in case series, either by including patients showing almost normal spermatogenesis, e.g. mild-to-moderate hypospermatogenesis, or by inclusion of patients with no testicular histology available. As a result of the above, retrieval rates reported in the literature for NOA men may vary from about 30% to even more than 80%. Larger case studies in well-defined NOA populations report sperm recovery rates after a first TESE attempt around 50%. However, given the invasive character of TESE, NOA patients want to have a better prediction of their chances to retrieve testicular sperm than tossing a coin. Because testicular volume and serum FSH are routinely assessed in azoospermic men, these parameters are often used in studies on prediction either as a stand-alone parameter or in combination with other parameters. Unfortunately, their predictive power remains limited and is subject to the heterogeneity of the population of NOA patients studied. Idem dito for the predictive parameters published in the study by Boitrelle et al. (2011). The positive likelihood ratios for the stand-alone parameters are below 2 and hence not of a great diagnostic value in predicting testicular sperm recovery. With a positive likelihood ratio of 3, a predictive score combining testicular volume, FSH and inhibin-B looks more promising in their setting. But again, is this a robust predictive model applicable to every population of NOA men? All NOA men with all parameters included in the score available were selected in our Brussels database. Although we refrained from assessing inhibin-B routinely, after concluding that this parameter had a poor predictive value (Vernaeve et al., 2002), the score could be calculated in a population of 110 NOA men that had their first TESE attempt and showed neither normal spermatogenesis nor hypospermatogenesis on their testicular histologies. Compared with the study by Boitrelle et al. (2011), our sperm retrieval rates were, respectively, 50.0% compared with 77.4% in men with a score below 18.5 (n 1⁄4 38) and 57.1% compared with 39.7% in men with a score between 18.5 and 3700 (n 1⁄4 72). Our ‘test’ population did not include men with a score higher than 3700. This ‘test’ emphasizes that any blind application of a prediction model on a population of patients, other than the one it originated from, remains difficult due to patient heterogeneity. Whether out-of-routine assessmentsmay improve the prediction of a successful testicular recovery also remains to be proven. Doppler ultrasoundor even invasive assessments such as ‘testicularmapping’ by puncture provide low sensitivities. Since many NOA men do not accept donor insemination as an alternative for their fatherhood and because current predictive tests cannot exclude that any sperm will eventually be retrieved, to date, reliable counselling towards sperm retrieval remains problematic for all TESE candidates suffering from NOA. Counselling becomes even more difficult when an NOA patient would like to know what his chances are to have a child, which in the end is the true outcome of interest to candidate TESE–ICSI patients. At present, only fragmentary data exist either reporting on retrieval rates after TESE or reporting on the outcome of ICSI once testicular spermatozoa were obtained in different subsets of patients. Boitrelle et al. (2011) show that in their setting, the take-home baby rate in patients with a score over 3700 was low (7.7%) compared with lower-score patients (48.8%), a finding that still may suffer from bias because only a subset of patients was included in the subsequent ICSI analysis (89 out of 149 men in which testicular sperm were retrieved, i.e. 59%, eventually proceeded to ICSI). Again, the score can probably not be applied blindly in other TESE–ICSI programmes

Can a machine-learning-based model trained in clinical and biological variables support the prediction of the presence or absence of sperm in testicular biopsy in non-obstructive azoospermia (NOA) patients? Our machine-learning model was able to accurately predict (AUC of 0.8) the presence or absence of spermatozoa in patients with NOA. Patients with NOA can conceive with their own biological gametes using ICSI in combination with successful testicular sperm extraction (TESE). Testicular sperm retrieval is successful in up to 50% of men with NOA. However, to the best of our knowledge, there is no existing model that can accurately predict the success of sperm retrieval in TESE. Moreover, machine-learning has never been used for this purpose. A retrospective cohort study of 119 patients who underwent TESE in a single IVF unit between 1995 and 2017 was conducted. All patients with NOA who underwent TESE during their fertility treatments were included. The development of gradient-boosted trees (GBTs) aimed to predict the presence or absence of spermatozoa in patients with NOA. The accuracy of these GBTs was then compared to a similar multivariate logistic regression model (MvLRM). We employed univariate and multivariate binary logistic regression models to predict the probability of successful TESE using a dataset from a retrospective cohort. In addition, we examined various ensemble machine-learning models (GBT and random forest) and evaluated their predictive performance using the leave-one-out cross-validation procedure. A cutoff value for successful/unsuccessful TESE was calculated with receiver operating characteristic (ROC) curve analysis. ROC analysis resulted in an AUC of 0.807 ± 0.032 (95% CI 0.743-0.871) for the proposed GBTs and 0.75 ± 0.052 (95% CI 0.65-0.85) for the MvLRM for the prediction of presence or absence of spermatozoa in patients with NOA. The GBT approach and the MvLRM yielded a sensitivity of 91% vs. 97%, respectively, but the GBT approach has a specificity of 51% compared with 25% for the MvLRM. A total of 78 (65.3%) men with NOA experienced successful TESE. FSH, LH, testosterone, semen volume, age, BMI, ethnicity and testicular size on clinical evaluation were included in these models. This study is a retrospective cohort study, with all the associated inherent biases of such studies. This model was used only for TESE, since micro-TESE is not performed at our center. Machine-learning models may lay the foundation for a decision support system for clinicians together with their NOA patients concerning TESE. The findings of this study should be confirmed with further larger and prospective studies. The study was funded by the Division of Obstetrics and Gynecology, Soroka University Medical Center, there are no potential conflicts of interest for all authors.

STUDY QUESTION Can a machine-learning-based model trained in clinical and biological variables support the prediction of the presence or absence of sperm in testicular biopsy in non-obstructive azoospermia (NOA) patients? SUMMARY ANSWER Our machine-learning model was able to accurately predict (AUC of 0.8) the presence or absence of spermatozoa in patients with NOA. WHAT IS KNOWN ALREADY Patients with NOA can conceive with their own biological gametes using ICSI in combination with successful testicular sperm extraction (TESE). Testicular sperm retrieval is successful in up to 50% of men with NOA. However, to the best of our knowledge, there is no existing model that can accurately predict the success of sperm retrieval in TESE. Moreover, machine-learning has never been used for this purpose. STUDY DESIGN, SIZE, DURATION A retrospective cohort study of 119 patients who underwent TESE in a single IVF unit between 1995 and 2017 was conducted. All patients with NOA who underwent TESE during their fertility treatments were included. The development of gradient-boosted trees (GBTs) aimed to predict the presence or absence of spermatozoa in patients with NOA. The accuracy of these GBTs was then compared to a similar multivariate logistic regression model (MvLRM). PARTICIPANTS/MATERIALS, SETTING, METHODS We employed univariate and multivariate binary logistic regression models to predict the probability of successful TESE using a dataset from a retrospective cohort. In addition, we examined various ensemble machine-learning models (GBT and random forest) and evaluated their predictive performance using the leave-one-out cross-validation procedure. A cutoff value for successful/unsuccessful TESE was calculated with receiver operating characteristic (ROC) curve analysis. MAIN RESULTS AND THE ROLE OF CHANCE ROC analysis resulted in an AUC of 0.807 ± 0.032 (95% CI 0.743-0.871) for the proposed GBTs and 0.75 ± 0.052 (95% CI 0.65-0.85) for the MvLRM for the prediction of presence or absence of spermatozoa in patients with NOA. The GBT approach and the MvLRM yielded a sensitivity of 91% vs. 97%, respectively, but the GBT approach has a specificity of 51% compared with 25% for the MvLRM. A total of 78 (65.3%) men with NOA experienced successful TESE. FSH, LH, testosterone, semen volume, age, BMI, ethnicity and testicular size on clinical evaluation were included in these models. LIMITATIONS, REASONS FOR CAUTION This study is a retrospective cohort study, with all the associated inherent biases of such studies. This model was used only for TESE, since micro-TESE is not performed at our center. WIDER IMPLICATIONS OF THE FINDINGS Machine-learning models may lay the foundation for a decision support system for clinicians together with their NOA patients concerning TESE. The findings of this study should be confirmed with further larger and prospective studies. STUDY FUNDING/COMPETING INTEREST(S) The study was funded by the Division of Obstetrics and Gynecology, Soroka University Medical Center, there are no potential conflicts of interest for all authors.

Study question Does testicular histology predict successful or unsuccessful TEsticular Sperm Extraction (TESE) in Non-Obstructive Azoospermia (NOA) patients? Summary answer Testicular histology failed to predict successful TESE in 1 of 3 NOA patients. What is known already The management of patients with Non-Obstructive Azoospermia (NOA) involves TEsticular Sperm Extraction (TESE) combined with IntraCytoplasmic Sperm Injection (ICSI). Sperm retrieval is successful in up to 50% of men with NOA; however, there is no single clinical finding or investigation that can accurately predict a positive outcome. Previous studies have concluded that testicular histology is the best predictor of a successful or unsuccessful TESE. Study design, size, duration This is a retrospective study of 525 patients who underwent TESE between January 2018 and December 2020 in Humanitas Fertility Center. Participants/materials, setting, methods The cohort was dived in five groups: 287 NOA, 95 necrozoospermia, 18 anejaculation, 1 testicular trauma and 124 Obstructive Azoospermia (OA) patients. Main results and the role of chance Sperm was retrieved and cryopreserved in 218/287 patients with NOA (75,95%), 55/95 in necrozoospermia (57,89%), 18/18 anejaculation (100%), 1/1 testicular trauma (100%) and 120/124 in OA patients (96,77%). Interesting that, when we compare sperm recovery data through TESE with the histological assessment (HA) of corresponding testicular biopsy, we found discordance against HA. In particular we retrieved spermatozoa in 218 NOA patients but in 74 of them HA failed to detect them (33,94%). Moreover HA failed to detect spermatozoa in 8/55 (14,54%) in case of necrozoospermia, 1/18 (5,55%) in anejaculation and 10/120 (8,33%) in OA patients. Limitations, reasons for caution Testis's heterogeneity is a limitation of the study. Wider implications of the findings We found a discordance when HA is compared with sperm recovery data of corresponding testicular biopsy. This data suggest that in case of testicular diagnostics histology in 33,94% of NOA cases there is a wrong diagnosis and 1 of 3 patients it doesn’t retrieve and cryopreserve spermatozoa when it could. Trial registration number N A

Proteomic Analysis of Testicular Interstitial Fluid in Men with Azoospermia

Study question To investigate the value of anti-Müllerian hormone(AMH) determination for estimating the sperm retrieval rate(SRR) of microdissection testicular sperm extraction(micro-TESE) in non-obstructive azoospermia(NOA) patients. Summary answer NOA with low AMH would have more opportunity to present heterogeneous seminiferous tubules when micro-TESE was performed and had higher SRR, especially in idiopathic cases. What is known already For infertile patients with NOA, micro-TESE is considered to have higher SRR than traditional surgery methods. However, serum inhibin B, follicle-stimulating hormone (FSH) and various clinical parameters are not reliable predictors for the presence of focal spermatogenesis and SRR. In male, AMH is a glycoprotein secreted by Sertoli cells and facilitate the regression of Müllerian structures in the developing foetus. It is still controversial that whether AMH level has value to predict the SRR of micro-TESE. Study design, size, duration This was a retrospective case-control study. From September 2014 to May 2022, 502 NOA patients treated with micro-TESE were divided into different groups according to their surgery outcome and seminiferous tubules appearance. Age, testis volume, serum AMH, FSH and testosterone level were compared between the different groups. The differences of SRR and AMH level in NOA patients with different etiologies were also compared. Participants/materials, setting, methods Micro-TESE was performed at x10 to x20 magnification. An attempt was made to identify seminiferous tubules that were larger and more opaque than others. The procedure was terminated when sperm were retrieved. If all tubules were seen to have an identical appearance, at least three samples (upper, middle, and lower) were extracted. Venous blood samples were drawn from each patient (7–10 AM) after an overnight fast. FSH and AMH were measured by electrochemiluminescence immunoassay. Main results and the role of chance Testicular sperms were successfully retrieved in 270 cases (SRR=53.8%). There were no statistical differences in age, testicular volume, FSH and testosterone levels between the patients who succeeded and failed to obtain sperm (all P>0. 05). The patients who obtained sperms had lower serum AMH level than those without sperm [0.81(0.16, 3.26)μg/L vs.1.37(0.21, 4.84)μg/L, P<0.05]. Patients with orchitis or AZFc deletion, cryptorchidism, KS, idiopathic azoospermia would have different AMH level [0.15(0.01, 0.41)μg/L, 5.71(3.57, 8.26)μg/L, 2.29(1.36, 3.81)μg/L, 0.15(0.05, 0.39)μg/L, 2.46(0.75, 5.49)μg/L, P<0.05]. Idiopathic azoospermia patients who obtained sperms had higher age but lower testosterone and AMH level than those without sperm [(35.2±8.9) years vs. (32.5±5.5) years, P<0.05, (3.1±1.4)μg/L vs. (3.7±2.1)μg/L, P<0.05; 1.63(0.35, 3.84)μg/L vs.3.00(1.20, 6.68)μg/L, P<0.05], there were no statistical differences in testicular volume and FSH level between the two groups (P>0.05). Receiver operating characteristic (ROC) curve showed that cut-off of serum AMH for successful sperm retrieval of idiopathic azoospermia patients was determined to be 2.96, with a sensitivity of 0.710 and specificity of 0.523, area under the curve (AUC) was 0.649. In the cases presenting heterogeneous seminiferous tubules during micro-TESE had lower AMH level and higher SRR than those presenting homogeneous seminiferous tubules [0.55(0.12, 2.05)μg/L vs.2.99(0.76, 6.11)μg/L, 75.9%(236/311)vs. 17.8%(34/191), all P<0.05]. Limitations, reasons for caution Pathology analysis should be involved in the following study. Randomized controlled trial comparing micro-TESE and traditional TESE would demonstrate that whether idiopathic azoospermia patients with higher serum AMH level would have less benefit by microsurgery than patients with lower AMH level. Wider implications of the findings Recently, in our pathologic research, NOA patients with extremely lower serum AMH level were observed to have more opportunity to present severe hyalinization in their seminiferous tubules. Tubules with severe hyalinization have less Sertoli cells and seem very thin. Therefore, tubules with spermatogenesis would be easy to identify during micro-TESE. Trial registration number not applicable

The Value of Semen Flow-Cytometry Analysis in Patients With Non-Obstructive Azoospermia Before Considering Testicular Sperm Retrieval