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Purpose of Review: This article aims to describe new developments in the field of nonobstructive azoospermia biology, diagnostics, biomarkers, and therapeutic strategies., Recent Findings: Recent studies have investigated the molecular underpinnings of cellular dysfunction that is contributing to spermatogenic dysfunction and findings suggest abnormalities across both somatic and germ cells. Biomarkers to predict the chances of sperm retrieval are being explored utilizing cell free (cf) DNA and RNA from various body fluids, in addition to a full range of transcripts and epigenetics within seminal fluid. Various approaches are being explored to optimize sperm identification from surgical specimens including microfluidic and machine learning approaches. Finally, approaches to regenerating sperm production from males with nonobstructive azoospermia are evolving to include various 3-dimensional culture techniques with integration of computational modeling., Summary: The landscape of nonobstructive azoospermia biomarkers, molecular underpinnings, technological approaches to more reliably identify sperm and novel regenerative therapeutic strategies are likely to transform the field of male reproduction in years to come., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Male factor infertility affects 50% of infertile couples worldwide; the most severe form, non-obstructive azoospermia (NOA), affects 10-15% of infertile males. Treatment for individuals with NOA is limited to microsurgical sperm extraction paired with in vitro fertilization intracytoplasmic sperm injection. Unfortunately, spermatozoa are only retrieved in ~50% of patients, resulting in live birth rates of 21-46%. Regenerative therapies could provide a solution; however, understanding the cell-type-specific mechanisms of cellular dysfunction is a fundamental necessity to develop precision medicine strategies that could overcome these abnormalities and promote regeneration of spermatogenesis. A number of mechanisms of cellular dysfunction have been elucidated in NOA testicular cells. These mechanisms include abnormalities in both somatic cells and germ cells in NOA testes, such as somatic cell immaturity, aberrant growth factor signalling, increased inflammation, increased apoptosis and abnormal extracellular matrix regulation. Future cell-type-specific investigations in identifying modulators of cellular transcription and translation will be key to understanding upstream dysregulation, and these studies will require development of in vitro models to functionally interrogate spermatogenic niche dysfunction in both somatic and germ cells., (© 2023. Springer Nature Limited.)

Objective: To develop a machine learning algorithm to detect rare human sperm in semen and microsurgical testicular sperm extraction (microTESE) samples using bright-field (BF) microscopy for nonobstructive azoospermia patients., Design: Spermatozoa were collected from fertile men. Testis biopsies were collected from microTESE samples determined to be clinically negative for sperm. A convolutional neural network based on the U-Net architecture was trained using 35,761 BF image patches with fluorescent ground truth image pairs to segment sperm. The algorithm was validated using 7,663 image patches. The algorithm was tested using 7,663 image patches containing abundant sperm, as well as 7,985 image patches containing rare sperm., Setting: In vitro fertilization center and university laboratories., Patient(s): Normospermic and nonobstructive azoospermia patients., Intervention(s): None., Main Outcome Measure(s): Precision (positive predictive value [PPV]), recall (sensitivity), and F1-score of detected sperm locations., Result(s): For sperm-only samples, our algorithm achieved 91% PPV, 95.8% sensitivity, and 93.3% F1-score at ×10 magnification. For dissociated microTESE samples doped with an abundant quantity of sperm, our algorithm achieved 84.0% PPV, 72.7% sensitivity, and 77.9% F1-score. For dissociated microTESE samples doped with rare sperm, our algorithm achieved 84.4% PPV, 86.1% sensitivity, and 85.2% F1-score., Conclusion(s): Rare sperm can be detected in patients' testis biopsy samples for potential subsequent use in in vitro fertilization-intracytoplasmic sperm injection. A machine learning algorithm can use BF images at ×10 magnification to accurately detect sperm locations using automated imaging., (Copyright © 2022 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To study the feasibility and spermatogenic potential of 3-dimensional (3D) bioprinting personalized human testicular cells derived from a patient with nonobstructive azoospermia (NOA)., Design: A human testicular biopsy from a single donor with NOA was dissociated into single cells, expanded in vitro, and 3D bioprinted into tubular structures akin to the seminiferous tubule using AGC-10 bioink and an RX1 bioprinter with a CENTRA coaxial microfluidic printhead from Aspect Biosystems. Three-dimensional organoid cultures were used as a nonbioprinted in vitro control., Setting: Academic medical center., Patient(s): A 31-year-old man with NOA with testis biopsy demonstrating Sertoli cell-only syndrome., Intervention(s): Three-dimensional bioprinting and in vitro culturing of patient-derived testis cells., Main Outcome Measure(s): Cellular viability after printing was determined, along with the expression of phenotypic and spermatogenic functional genetic markers after 12 days of in vitro culture., Result(s): Testicular cultures were expandable in vitro and generated sufficiently large numbers for 3D bioprinting at 35 million cells per mL of bioink. Viability 24 hours after printing was determined to be 93.4% ± 2.4%. Immunofluorescence staining for the phenotype markers SRY-Box transcription factor 9, insulin-like 3, actin alpha 2 smooth muscle, and synaptonemal complex protein 3 after 12 days was positive, confirming the presence of Sertoli, Leydig, peritubular myoid, and meiotic germ cells. Reverse transcription qualitative polymerase chain reaction analysis showed that after 12 days in spermatogenic media, the bioprints substantially up-regulated spermatogenic gene expression on par with nonbioprinted controls and showed a particularly significant improvement in genes involved in spermatogonial stem cell maintenance: inhibitor of deoxyribonucleic acid binding 4 by 365-fold; fibroblast growth factor 3 by 94,152-fold; stem cell growth factor receptor KIT by twofold; stimulated by retinoic acid 8 by 125-fold; deleted in azoospermia-like by 114-fold; synaptonemal complex protein 3 by sevenfold; zona pellucida binding protein by twofold; transition protein 1 by 2,908-fold; and protamine 2 by 11-fold., Conclusion(s): This study demonstrates for the first time the feasibility of 3D bioprinting adult human testicular cells. We show that the bioprinting process is compatible with high testicular cell viability and without loss of the main somatic phenotypes within the testis tissue. We demonstrate an increase in germ cell markers in the 3D bioprinted tubules after 12 days of in vitro culture. This platform may carry future potential for disease modeling and regenerative opportunities in a personalized medicine framework., (Copyright © 2022 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Purpose: We sought to determine if testicular histopathological heterogeneity is associated with sperm retrieval rates (SRRs) in men with nonobstructive azoospermia (NOA) who are undergoing microdissection testicular sperm extraction (mTESE)., Materials and Methods: All patients undergoing mTESE by a single, high-volume surgeon at a tertiary infertility referral center between 2010 and 2020 were evaluated. Pathology reports from testis biopsy at the time of mTESE reported by fellowship-trained genitourinary pathologists were reviewed. Testicular heterogeneity was correlated to absolute SRRs. Logistic regression was used to determine if heterogeneity was associated with sperm retrieval., Results: A total of 918 men with mTESE were included. Of these, 391 men (43%) had 1 pathology, 388 men (42%) had 2, 108 (12%) had 3, and 31 (3.4%) had 4. Overall, the most common histopathology was Sertoli-cell only, followed by maturation arrest. The overall SRR was 42% with a clinical intrauterine gestation rate of 30%. Increasing histopathological variety was associated with higher SRRs (p <0.01); a SRR of 33% was observed when one histopathological subtype was present vs 94% with 4 subtypes. Furthermore, men with any foci of spermatogenesis had higher SRRs., Conclusions: In men with NOA, increasing testicular histopathological heterogeneity is correlated with higher SRRs driven by the identification of focal areas of spermatogenesis. This is an important, although predictable, observation. While diagnostic biopsy is not routinely required, these findings emphasize the value of having histology to perhaps predict the chance of sperm retrieval for future mTESE procedures.

Objective: To assess the timing of patency and late failure (secondary azoospermia) after vasovasostomy (VV) using standardized kinetics definitions., Design: Retrospective cohort study., Setting: University-affiliated hospital., Patient(s): Patients with obstructive azoospermia., Intervention(s): Vasovasostomy., Main Outcome Measure(s): Univariate and multivariate logistic regression assessed predictors of patency and late failure. Patency was defined as any sperm return to the ejaculate; and >2 million total motile sperm (TMS) in ejaculate. Late failure after VV was defined as azoospermia; or <2 million TMS in ejaculate., Result(s): 429 men underwent VV, with median follow up of 242 days. Mean time to patency was 3.25 months versus 5.29 months in the "any sperm" versus ">2 million TMS" groups. Finding sperm intraoperatively during VV significantly improved patency rates in multivariable analysis (odds ratio [OR] 4.22). This association was further boosted when sperm was found bilaterally (OR 6.70). Late failure rate (azoospermia) was 10.6% at mean time of 14.1 months and 23% for <2 million, at mean time of 15.7 months. When assessing predictors of late failure, intraoperative motile sperm bilaterally was a statistically significant protective factor on multivariate analysis (hazard ratio 0.22)., Conclusion(s): Vasovasostomy remains highly efficacious in treating obstructive azoospermia. Young patients, shorter obstructive intervals, and sperm identified intraoperatively predict improved outcomes. Clinicians can expect VV patency in 3 months and late failure within the first 2 years after surgery. However, patency rates, late failure rates, and kinetics vary by definition., (Copyright © 2019 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)