Successful Microdissection Testicular Sperm Extraction Research Articles

P-570 A cell-free DNA test in semen of nonobstructive azoospermic men to predict spermatogenesis

Abstract Study question Can a genetic assessment of cell-free DNA, isolated from seminal plasma of nonobstructive azoospermic (NOA) men, predict successful sperm extraction at testicular biopsy? Summary answer Seminal cell-free DNA (cfDNA) isolated from the ejaculates of NOA men with failed testicular sperm retrieval outcomes concurrently and consistently displayed mutations on three genes. What is known already The most puzzling form of azoospermia is the testicular type, where the chances of successful microdissection testicular sperm extraction (micro-TESE) are unpredictable. Therefore, developing a method that could identify or predict individuals with absent spermatogenesis would be most useful. While histopathology is considered the most reliable method for predicting successful micro-TESE, it is invasive and often inconclusive. Recently, cfDNA testing has been gaining momentum as a noninvasive method for prenatal screening and even with the potential for eventually replacing PGT-A. However, the prognostic value of using cfDNA to predict the presence/absence of spermatozoa in men undergoing micro-TESE is relatively unknown. Study design, size, duration Over a period of 8 months, we recruited azoospermic men with normal peripheral karyotypes and for whom an extensive semen analysis did not identify spermatozoa. For consenting patients (n = 12), we performed genetic assessments on cfDNA from their ejaculates (IRB 1006101085). These patients subsequently underwent micro-TESE, and their cfDNA mutation profiles were compared according to whether spermatozoa were successfully retrieved ((+)Sperm, n = 5), or not ((-)Sperm, n = 7). Participants/materials, setting, methods Using a commercially available kit, circulating cfDNA was eluted from the ejaculates of consenting NOA men, as well as a normozoospermic control (n = 2). The isolated cfDNA specimens subsequently underwent whole exome sequencing (WES) on an Illumina HiSeq at 2x150bp. Gene mutations were detected by CLC Genomic Server 9.0 and compared within, as well as between, the +Sperm and -Sperm cohorts in comparison to the control group. Main results and the role of chance All 12 men (35.5±4yrs) had normal peripheral karyotypes and tested negative for Y-microdeletions. They had no history of genetic diseases and were off any supplements, medications, and/or hormones before or during the study. Genetic assessment on cfDNA from the +Sperm cohort (n = 5; 38.3±2yrs) identified 8 genes (ADAMTS7, ATP9B, CBFA2T3, FAM20C, FCGBP, PEBP4, PHF2, RPS9) that were concurrently mutated among all NOA men from this group. Although ADAMTS7 plays a potential role in sperm-egg fusion, the remaining genes are unrelated to sperm production. Whole exome sequencing of cfDNA from the -Sperm cohort (n = 7; 33.5±4yrs) consistently detected mutations on 3 genes, including one involved in spermatogenesis (CFAP54), one essential for oxidative regulation of spermatozoa (GSTT4), and one that encodes testis antigens that are normally expressed during spermatogenesis (BAGE2). Moreover, we concurrently and consistently identified pathogenic mutations on an additional gene in the -Sperm cohort. This gene (MUC2) is involved in the epithelial coating of mucus membrane-containing organs, and its expression has been correlated with spermatogenesis. Interestingly, these genes were all unaffected in men with successful micro-TESE outcomes. Limitations, reasons for caution Although we detected candidate gene mutations that correlate with micro-TESE outcome, these are preliminary findings that need further validation in a larger study cohort. Furthermore, while cfDNA analysis has shown great promise, it is not specific enough on its own and should be corroborated by epigenetic testing. Wider implications of the findings Genetic screening of seminal cfDNA provides a noninvasive alternative to histopathology and can be used to predict the absence of spermatogenesis, sparing these men from unnecessary surgery and cost. This novel biomarker approach lays the groundwork for precision medicine in male infertility testing. Trial registration number n/a

A Prior History of Cryptozoospermia Is Associated with a Significantly Higher Chance of a Successful Microdissection Testicular Sperm Extraction Compared to Non-Obstructive Azoospermia

Background: Our study sought to evaluate the rates of successful sperm retrieval following microdissection testicular sperm extraction (mTESE) in patients with a prior history of cryptozoospermia, compared to patients with non-obstructive azoospermia (NOA). Methods: A retrospective chart analysis evaluating all mTESE procedures was performed from January 2004 to August 2018. Inclusion criteria involved all males >18 years of age with a diagnosis of cryptozoospermia and/or NOA that underwent a mTESE. The patient’s genetic profile, hormonal profile, semen analysis, testicular volumes, pathology and comorbidities were analyzed. Results: We identified 40 patients with cryptozoospermia and 221 patients with NOA. Successful mTESE occurred in 34/40 (85%) cryptozoospermic males compared to 104/221 (48%) NOA males (p < 0.001). In univariate and multivariate analyses, patients with cryptozoospermia were more likely to undergo a successful mTESE than patients with NOA (OR 5.56 [1.79–17.29], p = 0.003; OR 5.41 [1.94–15.08], p = 0.0013), respectively. Factors that were associated with a statistically significant lower chance of successful mTESE included Sertoli-cell only pathology, pre-operative testosterone < 300 ng/dL and FSH > 7.6 mIU/mL. Conclusion: Despite patients with a history of cryptozoospermia having a significantly higher chance of a successful sperm retrieval than patients with NOA, couples should be counselled on the possibility of an unsuccessful sperm extraction, in order to optimize the pre-operative IVF planning and to manage operative expectations.

Seminal plasma extracellular vesicles tRF-Val-AAC-010 can serve as a predictive factor of successful microdissection testicular sperm extraction in patients with non-obstructive azoospermia

BackgroundThere is a lack of biomarkers for distinguishing non-obstructive azoospermia (NOA) patients with successful sperm retrieval (Sp+) from those with failed sperm retrieval (Sp-). This study aimed to determine the potential of extracellular vesicles tRNA-derived small RNA (tsRNA) as a novel non-invasive biomarker for successful sperm retrieval by microdissection testicular sperm extraction (mTESE).MethodsThe study included 18 patients with NOA with successful sperm retrieval (Sp+) and 23 patients with NOA with failed sperm retrieval (Sp-), 15 obstructive azoospermia (OA) patients, 5 idiopathic oligospermia (IO) patients, and 12 healthy people. Seminal plasma extracellular vesicles tsRNA levels were used in a two-stage case-control study (screened by tsRNA sequencing on Illumina NextSeq instrument and validated by qRT-PCR). The bioinformatic analysis was performed to determine the role of tsRNA in the pathogenesis of non-obstructive azoospermia.ResultsTwo tsRNAs (tRF-Val-AAC-010: AUC = 0.96, specificity = 80%, sensitivity = 95%; tRF-Pro-AGG-003: AUC = 0.96, specificity = 87%, sensitivity = 95%) were found to have high predictive accuracy for distinguishing the origin of azoospermia. In addition, the extracellular vesicles tRF-Val-AAC-010 resulted in high predictive ability (AUC = 0.89, sensitivity = 72%, specificity = 91%, P < 0.0001) in predicting the presence of sperm in non-obstructive azoospermia undergoing mTESE. Finally, bioinformatic analysis revealed that tRF-Val-AAC-010 were involved in spermatogenesis.ConclusionsThis study identified that the extracellular vesicles tRF-Val-AAC-010 and tRF-Pro-AGG-003 are biomarkers for the diagnosis of non-obstructive azoospermia, and that tRF-Val-AAC-010 as a potential non-invasive biomarker for predicting the presence of sperm in non-obstructive azoospermia testicular tissue.

O-314 Epigenetic Profiling of Seminal Plasma in NOA Men to Predict Successful Testicular Sperm Retrieval

Abstract Study question Can epigenetic profiling of seminal plasma be used to predict successful testicular sperm retrieval for men with non-obstructive azoospermia (NOA)? Summary answer Epigenetic screening of cell-free seminal RNA identified gene imbalances in NOA men, with specific correlation to those who failed to yield spermatozoa at testicular biopsy. What is known already Although the chance of a successful microdissection testicular sperm extraction (micro-TESE) in men with NOA can be up to 60%, the procedure may still fail to yield spermatozoa. Several factors have been proposed to predict a successful retrieval, including FSH, inhibin B, genetics, and histopathology. Although histopathology would be the most reliable of these to predict successful micro-TESE, it is equally invasive to perform. Indeed, cell-free RNA extracted from testicular biopsy specimens has been shown to be differentially expressed in infertile men according to the origin of their azoospermia, whether obstructive or nonobstructive, and in relation to a normozoospermic control. Study design, size, duration Over a 2-year period, we identified men in whom no spermatozoa were identified despite extensive semen analyses conducted by multiple embryologists. These patients, who were negative for Y microdeletion, subsequently underwent micro-TESE. For consenting men, we performed epigenetic analyses on their seminal plasma by RNAseq. Significant differentially expressed gene (DEG) profiles were then assessed and compared according to whether surgical sperm retrieval successfully yielded spermatozoa (+TESE) or not (-TESE). Participants/materials, setting, methods RNA was isolated from the ejaculates for RNAseq using a commercially available spin column kit. RNA isolates were sequenced by Illumina HiSeq at 2x150bp. An absolute log2fold change of &amp;gt; 1 and a P-value of &amp;lt; 0.05 was considered significant. DEG profiles were compared within, as well as between, the +TESE and -TESE cohorts in comparison to a donor control. Main results and the role of chance All 12 men (37.3±6yrs) had normal peripheral karyotypes. Six (38.0±7yrs) underwent successful testicular sperm retrievals, defining the +TESE cohort. These men exclusively shared 10 significantly imbalanced genes involved in processes such as spermatogenesis (n = 4), sperm function (n = 2), and testis development (n = 1). For the 6 men (36.6±5 yrs) who underwent testicular sperm retrievals that failed to yield spermatozoa (-TESE), we identified 16 significantly imbalanced genes, exclusively shared by these patients. These genes are mainly involved in spermatogenesis (n = 9), sperm maturation (n = 1), and cell cycle regulation (n = 4). We then compared the DEG profiles between the +TESE and -TESE cohorts and identified 8 imbalanced genes that were shared among all 12 NOA men. Of interest, TPTE2 was partially (67%) expressed in patients from the +TESE group, while IGSF11-AS1 was underexpressed in all men from the -TESE group. Both of these genes are implicated in spermatogenic defects and are normally highly expressed in the testis. Interestingly, we identified a gene (NA) that was solely and specifically underexpressed in all men from the -TESE group, yet simultaneously overexpressed in all men from the +TESE group. NA, which is well known for its role in sialic acid metabolism, is also present on the sperm acrosome. Limitations, reasons for caution Using non-invasive RNAseq on the seminal plasma of NOA men, we were able to identify DEGs according to whether spermatozoa were successfully retrieved or had failed retrieval with micro-TESE. Although intriguing, these are preliminary results that should be further validated in a larger study cohort. Wider implications of the findings RNAseq identified genes shared within the same prognostic cohort. Moreover, differential expression of some specific genes predicted micro-TESE outcome. This epigenetic assessment, carried out on the ejaculate, can therefore be used as a non-invasive biomarker tool to predict loss of spermatogenesis in NOA men, sparing them from unnecessary surgery. Trial registration number N/A

Circulatory exosomal tRF-Glu-CTC-005 and tRF-Gly-GCC-002 serve as predictive factors of successful microdissection testicular sperm extraction in patients with nonobstructive azoospermia

To identify circulating plasma exosomal transfer RNA-derived fragments (tRFs) as the predictive factors of successful microdissection testicular sperm extraction (micro-TESE) in patients with nonobstructive azoospermia (NOA). Case and control prospective study. Academic research laboratory. Twelve patients with NOA with successful sperm retrieval by micro-TESE, 18 patients with NOA with failed sperm retrieval by micro-TESE, and 12 normozoospermic fertile controls. Blood samples were collected from participants. The abundance of tRFs normalized as counts per million of the total aligned reads with the next-generation sequencing system; candidate tRF levels were validated through quantitative reverse transcription polymerase chain reaction; predictive accuracy was evaluated by the receiver operating characteristic area under the curve analysis. The nomogram was built for ranking. The plasma circulating exosomal tRF-Gly-GCC-002 and tRF-Glu-CTC-005 manifested the most confident differential expression between patients with NOA with successful sperm retrieval by micro-TESE and patients with NOA with failed sperm retrieval by micro-TESE. The target gene prediction of these 2 tRFs followed by the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis indicated the functional enrichment of neuroendocrine protein metabolism and striatum/subpallium development. The herpes simplex virus 1 infection pathway was also involved. The receiver operating characteristic area under the curve (AUC) analysis demonstrated a promising predictive accuracy: tRF-Gly-GCC-002, AUC of 0.921, and tRF-Glu-CTC-005, AUC of 0.954. A regression model was built and presented with the nomogram for further assessment. This study described the exosomal tRF-Gly-GCC-002 and tRF-Glu-CTC-005 expression values, indicated a promising predictive effect for accessibility of sperm retrieval through micro-TESE from patients with NOA, and highlighted tRF-Gly-GCC-002 and tRF-Glu-CTC-005 as useful biomarkers in patients with NOA seeking invitro conception with their residual sperm.

Predictive factors of successful microdissection testicular sperm extraction in patients with presumed sertoli cell-only syndrome.

To evaluate predictive factors of successful microdissection-testicular sperm extraction (MD-TESE) in patients with presumed Sertoli cell-only syndrome (SCOS). In this retrospective analysis, 874 men with non-obstructive azoospermia (NOA), among whom 148 individuals with diagnosis of SCOS in prior biopsy, underwent MD-TESE at Department of Andrology, Royan Institute, Tehran, Iran. The predictive values of follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) levels, testicular volume, as well as male age for retrieving testicular sperm by MD-TESE were analyzed by multiple logistic regression analysis. Testicular sperm were successfully retrieved in 23.6% men with presumed SCOS. Using receiver operating characteristic (ROC) curve analysis, it was shown that sperm retrieval rate in the group of men with FSH values >15.25% was 28.9%. This was higher than the group of men with FSH ≤15.25 (11.8%). Sperm retrieval rate (SRR) was 23.6% in men with presumed SCOS and FSH level can be a fair predictor for SPR at MD-TESE. MD-TESE appears to be recommendable in such cases (SCOS with high FSH concentration) with reasonable results.

Predictive factors of successful microdissection testicular sperm extraction.

Azoospermia in men requires microsurgical reconstruction or a procedure for sperm retrieval with assisted reproduction to allow fertility. While the chance of successful retrieval of sperm in men with obstructive azoospermia approaches >90%, the chances of sperm retrieval in men with non-obstructive azoospermia (NOA) are not as high. Conventional procedures such as fine needle aspiration of the testis, testicular biopsy and testicular sperm extraction are successful in 20-45% of men with NOA. With microdissection testicular sperm extraction (micro-TESE), the chance of successful retrieval can be up to 60%. Despite this increased success, the ability to counsel patients preoperatively on their probability of successful sperm retrieval has remained challenging. A combination of variables such as age, serum FSH and inhibin B levels, testicular size, genetic analysis, history of Klinefelter syndrome, history of cryptorchidism or varicocele and histopathology on diagnostic biopsy have provided some insight into the chance of successful sperm retrieval in men with NOA. The goal of this review was to evaluate the preoperative factors that are currently available to predict the outcome for success with micro-TESE.

Successful Repeat Microdissection Testicular Sperm Extraction in Men With Nonobstructive Azoospermia

We studied factors that can predict successful repeat microdissection testicular sperm extraction in men with nonobstructive azoospermia. We retrospectively analyzed the records of 126 men with nonobstructive azoospermia who underwent 1 successful microdissection testicular sperm extraction attempt. Clinical factors identifiable at the second procedure, including age, testicular volume, endocrinological data and histology, were analyzed. Overall testicular spermatozoa were successfully retrieved at 103 of 126 repeat attempts (82%). Men with a successful repeat attempt had lower follicle-stimulating hormone (mean±SD 23.1±12.4 vs 29.2±12.8, p=0.04) and larger testicular volume (mean 10±5 vs 7±4, p=0.0001) at the repeat procedure compared to men with a failed repeat attempt. Adjusted associations from a multiple logistic regression model showed that no factors predicted sperm retrieval during repeat microdissection testicular sperm extraction. An ROC curve showed a fair prediction model (AUC=0.71). The follicle-stimulating hormone level and testicular volume at the repeat attempt appear to have predictive value to determine the success of a second attempt. These observations are interesting since testicular volume and follicle-stimulating hormone in men with nonobstructive azoospermia do not predict sperm retrieval at a primary microdissection testicular sperm extraction attempt.

Successful Fertility Treatment for Klinefelter's Syndrome

We examined preoperative factors that could predict successful microdissection testicular sperm extraction in men with azoospermia and nonmosaic Klinefelter's syndrome. We also analyzed the influence of preoperative hormonal therapy on the sperm retrieval rate. A total of 91 microdissection testicular sperm extraction attempts were done in 68 men with nonmosaic Klinefelter's syndrome. Men with serum testosterone less than 300 ng/dl received medical therapy with aromatase inhibitors, clomiphene or human chorionic gonadotropin before microdissection testicular sperm extraction. Preoperative factors of patient age and endocrinological data were compared in those in whom the procedure was and was not successful. The sperm retrieval rate was the main outcome. Clinical pregnancy (pregnancy with heartbeat) and the live birth rate were also calculated. Testicular spermatozoa were successfully retrieved in 45 men (66%), representing 62 (68%) attempts. Increasing male age was associated with a trend toward a lower sperm retrieval rate (p = 0.05). The various types of preoperative hormonal therapies did not have different sperm retrieval rates but men with normal baseline testosterone had the best sperm retrieval rate of 86%. Patients who required medical therapy and responded to that treatment with a resultant testosterone of 250 ng/dl or higher had a higher sperm retrieval rate than men in whom posttreatment testosterone was less than 250 ng/dl (77% vs 55%). For in vitro fertilization attempts in which sperm were retrieved the clinical pregnancy and live birth rates were 57% and 45%, respectively. Microdissection testicular sperm extraction is an effective sperm retrieval technique in men with Klinefelter's syndrome. Men with hypogonadism who respond to medical therapy may have a better chance of sperm retrieval.

Sensitivity, Specificity and Accuracy of Intraoperative Findings in Microdissection Testicular Sperm Extraction: A Prospective Study

Background: Identification of healthy looking testicular tissue is the rationale behind successful microdissection testicular sperm extraction (TESE). Objective: To determine sensitivity, specificity and accuracy of intraoperative microscopic findings during microdissection TESE as correlated to successful sperm retrieval. Patients and Methods: A consecutive 100 primary infertile men with non-obstructive azoospermia were prospectively enrolled for microdissection TESE in a single tertiary academic center. Under anesthesia, testicular parenchyma was examined at high magnification to identify dilated, opaque or whitish tubules which are considered to contain spermatozoa. Additionally 2 samples were taken from the adjacent tissues of non-dilated tubules for sperm retrieval and histological examination. If no dilated tubules were observed, multiple samples were randomly excised from middle and polar areas. Results: The mean age was 36.4 ± 8.4 years and the mean duration of infertility was 9.1 ± 7.2 years. Both testes were explored in 68 patients and one was examined in 32. Microdissection TESE retrieved mature spermatozoa in 33 patients (33%). A total of 168 testes were examined and the foci of healthy looking testicular tissue were identified in 44 (26.2%). A false positive result (dilated tubules with no sperms retrieved) was encountered in 10 cases. A false negative result (no dilated tubules were identified in spite of retrieval of mature sperms) was found in 3 cases. The over all sensitivity, specificity and accuracy of the examined testes were 91.9, 92.4 and 92.3 %, respectively. Hypospermatogenesis had the highest sperm retrieval rate. Sperm retrieval rate was not related to serum follicle-stimulating hormone, testosterone or previous sperm retrieval interventions but to the most advanced pattern of testicular histology (p = 0.027). Conclusion: Identification of healthy looking testicular tissue during microdissection TESE is highly sensitive, specific and accurate for successful sperm retrieval. Our results prove the rationale behind the procedure.

Clinical Comparison of Successful and Failed Microdissection Testicular Sperm Extraction in Patients with Nonmosaic Klinefelter Syndrome

Testicular sperm extraction (TESE), together with intracytoplasmic sperm injection, has become the treatment of choice for patients with nonobstructive azoospermia related to nonmosaic Klinefelter syndrome (KS) who want to father children. However, predicting successful microdissection TESE for patients with KS remains controversial. Thus, we conducted a study to determine whether the prediction of successful microdissection TESE is possible in patients with nonmosaic KS. The subjects were 26 men with nonmosaic KS who had undergone microdissection TESE. The patients were divided into two groups: those in whom the procedure was successful and those in whom it was not. Several factors, including patient age, testicular volume, endocrinologic data, and intraoperative morphologic appearance of the testes were compared between the two groups. Testicular spermatozoa were retrieved successfully in 13 (50%) of the 26 patients. None of the factors investigated differed significantly between the two groups according to the Mann-Whitney U test or multivariate logistic regression analysis. Testicular spermatozoa were successfully retrieved from 16 (94.1%) of the 17 testes in which the seminiferous tubules without sclerotic changes were observed but were not found in any of the 29 testes without seminiferous tubules (P <0.0001). For patients with nonmosaic KS, it is encouraging that testicular sperm were successfully identified in 50% of our patients. Although we did not find any factor predictive of successful testicular sperm retrieval by microdissection TESE, the outcome of microdissection TESE for patients with nonmosaic KS appears to depend on the identification of seminiferous tubules without sclerotic changes in the testicular tissue.

Four pregnancies in nonmosaic Klinefelter’s syndrome using cryopreserved-thawed testicular spermatozoa

To investigate feasibility of using cryopreserved-thawed testicular spermatozoa from patients with nonmosaic Klinefelter's syndrome for intracytoplasmic sperm injection (ICSI). Case report. University-based hospital and IVF clinic. Six patients with nonmosaic Klinefelter's syndrome who underwent testicular sperm extraction for ICSI. Microdissection testicular sperm extraction (TESE) and ICSI. We compared results of ICSI using cryopreserved testicular spermatozoa with those previously reported in Klinefelter's syndrome and those in nonobstructive azoospermia patients using cryopreserved testicular spermatozoa at our institution with respect to embryo cleavage rate, implantation rate, and pregnancy outcome. Four of six patient couples with successful microdissection TESE achieved pregnancy using cryopreserved-thawed testicular spermatozoa. One pregnancy resulted in early-pregnancy abortion, two in delivery of healthy singleton girls, and one delivery of a healthy singleton boy. Cryopreserved-thawed testicular spermatozoa can be used successfully for ICSI in patients with nonmosaic Klinefelter's syndrome.

Impact of age, follicle stimulating hormone and Johnsen's score on successful sperm retrieval by microdissection testicular sperm extraction.

Background: The spermatozoa retrieval rate achieved by microdissection testicular sperm extraction (TESE) for patients with non-obstructive azoospermia is still approximately 50%, despite technical advances. Predicting a successful outcome is important to avoid unnecessary surgery. A study was conducted to determine a method for calculating the predicted probability of successful microdissection TESE. Methods: Testicular spermatozoa were retrieved in 41 of 100 patients by microdissection TESE. Ten clinical factors were examined in the search for an optimal logistic model for predicting a good outcome. Once the best delimiting value was established, sensitivity and specificity were calculated. Results: Patient age, serum follicle stimulating hormone (FSH) concentration and Johnsen's score (JS) were shown to be influential factors by multivariate logistic analysis. The mathematical model for predicting the probability of a successful outcome was as follows: P=(1+exp[0.144×patient age-0.059×serum FSH concentration-1.310×JS])-1. When a predictive probability of 49.7% was selected as the best cut-off value, sensitivity and specificity were 78.0% and 76.3%, respectively. Conclusion: This present model is very useful for predicting successful microdissection TESE. In addition, we suggest that a younger patient age would increase the probability of success with this procedure. (Reprod Med Biol 2005; 4: 53-57).

Impact of age, follicle stimulating hormone and Johnsen’s score on successful sperm retrieval by microdissection testicular sperm extraction

Background The spermatozoa retrieval rate achieved by microdissection testicular sperm extraction (TESE) for patients with non-obstructive azoospermia is still approximately 50%, despite technical advances. Predicting a successful outcome is important to avoid unnecessary surgery. A study was conducted to determine a method for calculating the predicted probability of successful microdissection TESE.