Primate-specific genes (PSGs), important contributors to the origin of adaptive evolutionary novelties, are abundantly expressed in the testis. However, the specific roles of PSGs in the male reproductive system of humans and other primates are largely unknown. Here, we employed whole-exome sequencing and identified deleterious variants of TFDP3, an X-linked PSG, in eight infertile men with oligoasthenoteratozoospermia. All of the male subjects harboring TFDP3 variants presented dramatic reductions in sperm concentration, motility, and abnormal sperm morphology. Furthermore, Tfdp3-knockdown (KD) in the testes of cynomolgus monkeys confirmed the important role of TFDP3 in normal spermatogenesis in primates. Consistently, dramatic decreases in sperm concentration, motility, and abnormal sperm morphology were also observed in Tfdp3-KD male cynomolgus monkeys. More importantly, further functional studies revealed that TFDP3 deficiency activated E2F1 induced apoptosis and thus led to decreased sperm count and motility. In addition, five of the eight couples underwent intra-cytoplasmic sperm injection treatment and achieved a successful pregnancy, indicating a potentially good outcome of assisted reproduction for those with TFDP3 deficiency-mediated oligoasthenoteratozoospermia. Collectively, our genetic analyses and experimental observations in humans and cynomolgus monkeys highlight the crucial role of TFDP3, an inhibitor of apoptosis, in normal spermatogenesis. These findings expand the spectrum of pathogenic variants for oligoasthenoteratozoospermia-associated male infertility and also reveal the special significance of primate-specific TFDP3 for the human male reproductive system, thus providing important guidance for genetic counseling and the clinical diagnosis of male infertility.

STUDY QUESTION:How can rare spermatozoa be identified efficiently during microsurgical testicular sperm extraction (micro-TESE)? SUMMARY ANSWER:An artificial intelligence (AI)-assisted system was developed to flag candidate rare spermatozoa in real time during micro-TESE, and it may support embryologists as a decision-support tool. WHAT IS KNOWN ALREADY:Patients with non-obstructive azoospermia (NOA) can obtain sperm for procreation through micro-TESE. During this procedure, sperm retrieval primarily relies on embryologists or laboratory technicians visually searching for sperm under a microscope, which is not only laborious and inherently subjective but also susceptible to errors. Although AI technology has been applied to identify trace amounts of sperm, existing models lack sufficient efficiency and true real-time performance. STUDY DESIGN SIZE DURATION:This study included model development followed by a single-centre clinical evaluation. An improved YOLO (You Only Look Once)-based rare sperm detection model, termed YOLOv11-RSD, was developed using microscopy data from 1165 surgical patients, comprising 1932 image samples containing a total of 5032 annotated sperm objects with confirmed identification. Clinical evaluation was performed between May 2024 and July 2025. Performance was assessed across confidence thresholds in obstructive azoospermia (OA) patients with normal spermatogenesis, and the system was then applied during micro-TESE in NOA patients and compared with routine embryologist assessment. PARTICIPANTS/MATERIALS SETTING METHODS:The model was developed using testicular sperm microscopy images collected at a single hospital. Real-time clinical feasibility was evaluated in 10 OA cases and 30 NOA cases. Embryologist assessment was used as the reference standard, and performance was assessed using PPV, sensitivity, F1-score, and 95% confidence intervals. Discordant AI-assisted detections were reviewed by embryologists in real time. MAIN RESULTS AND THE ROLE OF CHANCE:YOLOv11-RSD achieved real-time detection of candidate spermatozoa in microscopy images with high sensitivity and acceptable PPV under the selected operating threshold. Compared with baseline YOLOv11, YOLOv11-RSD showed improved overall detection performance across representative evaluation settings. In OA cases, the system achieved high sensitivity for sperm detection, reaching up to 96.7% across evaluated thresholds. During micro-TESE in NOA patients, at a confidence threshold of 0.50, positive predictive value (PPV), sensitivity, and F1-score were 80.58%, 96.11%, and 87.66%, respectively. The system highlighted candidate spermatozoa that were not identified during the initial manual assessment in six NOA cases, including two cases initially classified as sperm-negative; these findings were confirmed upon immediate re-review. Follow-up reproductive outcomes were available for six cases in which AI-assisted detection contributed to the search-and-confirmation workflow: embryo cleavage was achieved in all six cases, and three cases ultimately resulted in live births. Notably, among the two cases initially classified as sperm-negative, one case resulted in a singleton live birth. LARGE SCALE DATA:N/A. LIMITATIONS REASONS FOR CAUTION:This was a single-centre clinical evaluation with a limited clinical cohort. Although model inference was rapid, procedure-level efficiency was constrained by image acquisition and scanning logistics, and no definitive reduction in total procedure time was demonstrated. External multi-centre validation is required. WIDER IMPLICATIONS OF THE FINDINGS:AI-assisted sperm detection may support embryologists during micro-TESE by flagging candidate rare spermatozoa for rapid review. Further prospective multi-centre validation is required to determine whether this approach improves procedure-level efficiency or clinical outcomes. FUNDING:This work was supported by grants from National Natural Science Foundation of China (82301794), Shanghai Science and Technology Innovation Action Plan (24Y12800702), Natural Science Foundation of Shanghai (25ZR1401300), National Key Research and Development Program of China (2022YFC270300), China Jiliang University Research Grant (No. H251120), and Shanghai General Hospital Basic and Clinical Collaborative Research Program (JC202612). DISCLOSURES:The authors declare no competing interests.

Background: Evidence on low-intensity pulsed ultrasound (LIPUS) for severe erectile dysfunction, particularly regarding long-term outcomes, remains limited. We assessed short-term within-group changes after LIPUS and respondent-reported outcomes at 3 years. Materials and methods: In this single-arm prospective observational study, 52 men commenced an 8-session LIPUS course; 43 completed treatment and had paired baseline and 8-week data. The primary endpoint was change in International Index of Erectile Function–Erectile Function. Erection Hardness Score and other exploratory secondary outcomes were analyzed with false discovery rate control. Three-year outcomes were summarized for 20 telephone respondents, with extreme-case analyses for 23 nonrespondents. Results: Mean International Index of Erectile Function–Erectile Function increased from 6.98 to 14.53 (mean change, 7.56; 95% confidence interval, 5.38–9.73; p < 0.001); 22/43 participants met the ≥7-point minimum clinically important difference (51.2%; 95% confidence interval, 36.8%–65.4%), or 22/52 (42.3%) when 9 discontinuers were classified as nonresponders. Erection Hardness Score improved in 31/43 participants ( p < 0.001). No adverse events were recorded among 43 participants with available safety data. Among 20 three-year respondents, positive responses were 80.0% for Sexual Encounter Profile question 2, 65.0% for Sexual Encounter Profile question 3, 70.0% for perceived erectile function improvement, and 55.0% for sexual satisfaction; corresponding worst-case proportions among all 43 participants were 37.2%, 30.2%, 32.6%, and 25.6%. Conclusions: Low-intensity pulsed ultrasound was associated with short-term within-group improvement in erectile function; however, the uncontrolled design, incomplete safety ascertainment, and substantial long-term nonresponse preclude causal inference or conclusions about durable benefit. Randomized, sham-controlled trials using consistent objective endpoints are warranted.

Non-obstructive azoospermia (NOA) represents the most severe type of male infertility. The linker of nucleoskeleton and cytoskeleton (LINC) complex and the TERB1-TERB2-MAJIN (TTM) telomere-tethering complex are essential for meiotic chromosome dynamics; however, pathogenic variants in these pathways have rarely been characterized in human NOA. In this study, whole-exome sequencing (WES) identified novel bi-allelic variants in genes encoding components of the LINC and TTM complexes in patients with NOA, including compound heterozygous variants in KASH5 (NM_144688.5:c.149-1G>A; NM_144688.5:c.876+1G>A), homozygous variant in TERB1 (NM_001136505:c.1814G>A, p.Arg605Gln), compound heterozygous variants in TERB2 (NM_152448.3:c.39dup, p.Ser14Ter; NM_152448.3:c.522A>G, p.Thr174Thr), and homozygous splice-site variant in MAJIN (NM_001318808.2:c.219+1G>T). Testicular histology and immunofluorescence analyses concordantly demonstrated a shared phenotype of meiotic prophase I arrest and absence of post-meiotic germ cells in these patients. Minigene assays demonstrated that the splice-site variants in KASH5 (c.149-1G>A; c.876+1G>A), TERB2 (c.522A>G), and MAJIN (c.219+1G>T) induced aberrant exon-skipping events. The frameshift variant in TERB2 (c.39dup) resulted in complete loss of protein expression, whereas the missense variant of TERB1 (c.1814G>A) led to markedly reduced protein levels and disruption of the TRF1-binding domain via in silico structural modeling. Collectively, this study demonstrated that novel variants in LINC and TTM complexes of meiotic chromosome dynamics are associated with meiotic arrest and NOA, which expanded the mutational spectrum of LINC- and TTM-related genes and deepen our knowledge of the role of LINC- and TTM-complex in male fertility.

QuestionDoes letrozole improve semen-based outcomes in men with spermatogenic failure?FindingsIn this randomized clinical trial of 296 men with spermatogenic failure, 14.3% of participants treated with letrozole (2.5 mg daily) plus vitamins C and E achieved an upgrade in World Health Organization sperm concentration category at 3 months compared with 5.4% in those receiving vitamins C and E alone, a statistically significant difference.MeaningIn this study, letrozole improved sperm concentration categories among men with spermatogenic failure, potentially enabling less invasive reproductive management. This randomized clinical trial evaluates whether treatment with letrozole improves sperm concentration categories among men with spermatogenic failure in China. ImportanceSpermatogenic failure (SPGF) is a severe form of male infertility with limited evidence-based medical treatment options. Aromatase inhibitors represent a promising therapeutic strategy for SPGF, but high-quality evidence is lacking.ObjectiveTo evaluate the efficacy and safety of letrozole for improving sperm concentration categories in men with SPGF.Design, Setting, and ParticipantsThis multicenter, open-label, assessor-blinded randomized clinical trial was conducted at 10 male infertility centers in China from July 2023 to March 2024. Men with SPGF (nonobstructive azoospermia [NOA], cryptozoospermia, or severe oligozoospermia) were enrolled. Follow-up for the primary outcome was completed in June 2024. Data were analyzed from July 2024 to March 2025.InterventionsParticipants were randomized to receive letrozole (2.5 mg daily) plus vitamins C and E or vitamins C and E alone (control group) for 3 months.Main Outcomes and MeasuresThe primary outcome was World Health Organization Sperm Concentration Categories (WHO-SCC) upgrade rate at 3 months after randomization. The secondary outcomes were WHO-SCC grades, Dutch Society of Obstetrics and Gynecology Total Motile Sperm Count Categories (NVOG-TMSCC) upgrade rate, semen parameters, and reproductive hormone levels.ResultsAmong 296 participants (mean [SD] age, 30.2 [3.9] years; 218 [73.6%] with NOA; 147 randomized to the letrozole group and 149 to the control group), 247 (83.4%) completed the trial, and all 296 were included in the primary analysis. WHO-SCC upgrade rates were 14.3% (21 of 147 participants) with letrozole vs 5.4% (8 of 149 participants) with control (risk difference, 9.2% [95% CI, 2.5%-15.8%]; P = .01). Participants in the letrozole group had higher odds of achieving a better WHO-SCC grade than those in the control group (common odds ratio, 2.65 [95% CI, 1.28-5.47]; P = .008). The NVOG-TMSCC upgrade rate was also higher with letrozole. Letrozole significantly increased serum gonadotropins and testosterone while decreasing estradiol. There were no significant between-group differences in semen parameters. Decreased libido (18 [12.2%] vs 8 [5.4%]; P = .04) was more frequent with letrozole than with control.Conclusions and RelevanceIn this randomized clinical trial of men with spermatogenic failure, letrozole significantly improved sperm concentration categories and was well tolerated, supporting its use as an option to downstage infertility severity and potentially enable less invasive reproductive management.Trial Registrationchictr.org.cn Identifier: ChiCTR2300073861

RATIONALE AND OBJECTIVES:Contrast-enhanced ultrasound (CEUS) is an effective method for assessing varicocele (VC) hemodynamics; however, its value in predicting improvements in semen parameters after varicocelectomy remains unclear. This study investigated whether CEUS-based hemodynamic patterns could predict surgical outcomes and developed and validated a novel predictive model. MATERIALS AND METHODS:In this prospective cohort study, 187 patients with VC undergoing microscopic varicocelectomy were included. A total of 23 clinical, grayscale ultrasound, and CEUS-based hemodynamic parameters were collected. The primary outcome was semen parameter improvement (≥25% increase in total motile sperm count) six months postoperatively. Elastic net regression (ENR) selected key predictors, which were then included in a multivariate logistic regression model. The resulting nomogram's performance was evaluated using receiver operating characteristic curves, calibration, and decision curve analysis, and was internally validated using 1000 bootstrap resamples. RESULTS:A total of 118 patients achieved post-operative semen parameter improvement. From 23 candidate variables, ENR identified three independent predictors as follows: left and right CEUS hemodynamic patterns and follicle-stimulating hormone. The nomogram demonstrated excellent discrimination, with an area under the curve of 0.822 and good calibration (Hosmer-Lemeshow test, P = 0.405). Decision curve analysis confirmed the clinical utility of the model across a wide range of risk thresholds. CONCLUSION:CEUS-based pampiniform plexus hemodynamic patterns are independent predictors of post-operative semen parameter improvement in patients with VC. We successfully developed and validated a novel predictive nomogram. This study provides an effective, non-invasive tool for surgical decision-making and expands the clinical application of CEUS.

Using testicular sperm, men with azoospermia can father children. Sperm cryopreservation helps avoid the risk of cycle canceling because of the failure of testicular sperm retrieval. However, cryopreservation of testicular sperm can potentially impair sperm function. In addition, the different qualities of testicular sperm retrieved from men with various etiologies of azoospermia can lead to different intracytoplasmic sperm injection (ICSI) outcomes after sperm cryopreservation. A retrospective analysis was performed to elucidate the influence of frozen testicular sperm from men with different etiologies of azoospermia on assisted reproductive technology (ART) treatment outcomes. Testicular sperm retrieval rates were also compared. The fertilization rate was found to be influenced by testicular sperm cryopreservation in most cases, especially for patients with azoospermia factor c (AZFc) deletion, and embryo development was significantly impeded when frozen testicular sperm from Klinefelter syndrome (KS) patients were used. Moreover, ART treatment outcomes after embryo transfer were not significantly influenced by testicular sperm cryopreservation for any of the etiologies. In addition, significantly higher sperm retrieval rates were achieved in cryptorchidism, KS, and AZFc deletion patients, whereas the testicular sperm retrieval rate was significantly lower in patients with idiopathic nonobstructive azoospermia. Collectively, our findings suggest that except for patients with KS, testicular sperm retrieval prior to initiating ovarian stimulation of the patient's partner should be considered the first-line approach. In addition, assisted oocyte activation should be performed in ICSI cycles using frozen testicular sperm, especially for patients with AZFc deletion.

Background:Microdissection testicular sperm extraction (micro-TESE) is an important technique for retrieving sperm in cases of non-obstructive azoospermia (NOA). However, the success rate of sperm retrieval using micro-TESE remains suboptimal. This has created an urgent clinical need to identify reliable predictive indicators that can accurately determine which NOA patients are likely to benefit from the procedure. Current literature lacks comprehensive studies correlating non-invasive ultrasound-based testis parameters with micro-TESE outcomes, highlighting a gap in the ability to preoperatively predict surgical success. This study aimed to investigate potential preoperative predictors of surgical success. Methods:We retrospectively analyzed data from 167 men with azoospermia treated at our hospital between January 2020 and December 2024. We investigated the correlations between Johnsen score and various parameters, including testicular volume (TV), rete testis thickness (RTT), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T), using Spearman rank correlation analysis. Additionally, we employed binary logistic regression to evaluate the predictive ability of these parameters for micro-TESE outcomes. Results:Our findings revealed significant positive correlations of Johnsen score with age, RTT, and TV (r=0.136, 0.783, and 0.706, respectively), and negative correlations with FSH and LH (r=-0.723 and -0.515, respectively). Our binary logistic regression model was Logit(P)=-6.828 + 4.753 × RTT - (-0.059) × FSH + 0.061 × LH. The sensitivity, specificity, Youden index, and area under the curve (AUC) values for predicting the results of micro-TESE were 85.6%, 95.0%, 0.81, and 0.950 [95% confidence interval (CI): 0.927-0.974], respectively. Conclusions:RTT is strongly correlated with Johnsen score; a combination of RTT, TV, FSH, and LH can serve as a reliable preoperative predictor of micro-TESE outcomes. These findings may improve patient selection for micro-TESE, thereby enhancing the overall success rates of this procedure in NOA patients.

Uniform testicular maturation arrest is a severe form of male infertility characterized by the presence of germ cells that do not complete spermatogenic development. It is usually caused by meiotic arrest with genetic variants and is difficult to treat via drugs or surgery. mRNA-lipid nanoparticle (LNP) delivery is a promising therapeutic option for maturation arrest with monogenic variants via protein replacement therapy. Herein, a spermatocytes-tropic LNP (Pool1-LNP3) was identified via a library of 30 ionizable lipids screening. And in vivo delivery of this novel LNP composition using rete testis microinjection was shown to be high spermatocytes targeting with high transfection efficiency. Thereafter, it was revealed that in vivo delivery of Pool1-LNP3 encapsulating Msh5 mRNA could promote crossover formation and restore spermatogenesis in Msh5D486Y/D486Y mouse models with DNA double-strand break (DSB) recombination defects. Notably, the offspring without genomic integration was born using intracytoplasmic sperm injection (ICSI) derived from the rescue of Msh5D486Y/D486Y mouse and embryo transfer. In addition, it was demonstrated that Maps mRNA-LNP3 recovered spermatogenesis in Maps KO mouse with meiotic arrest. Altogether, these findings suggested that this spermatocyte-tropic mRNA-LNP delivery could become a viable and applicable strategy for the treatment of spermatogenic disorders with genetic defects, providing a foundation for future clinical application.

During meiosis, ZMM proteins play essential roles in stabilizing the recombination intermediates and promoting crossover (CO) formation. In mice, shortage in chiasmata 1 (SHOC1) forms a trimeric complex with the other two ZMM proteins, SPO16 and TEX11, to bind recombination intermediates after strand invasion. Although genetic variants of SHOC1 are clinically associated with male infertility, their conserved functions in human gametogenesis remain enigmatic. Here, we delineated species-specific divergences between human and mouse SHOC1 complex and identified a missense variant within the XPF-like domain in SHOC1 (p.Q590R). This variant impaired DNA double-strand breaks repair by compromising its ability to bind branched DNA structures and the recruitment of crucial proteins to recombination intermediates, ultimately abolishing CO formation. Furthermore, the variant disrupted dynamic chromatin structure in pachytene spermatocytes and induced synapsis defects. Importantly, the XPF-like domain in SHOC1 was revealed to prevent autosome intrusion into the sex body compartment, thereby protecting critical autosomal loci from meiotic silencing of unsynapsed chromatin (MSUC). Overall, our study underscores the critical role of the XPF-like domain in human SHOC1 in CO formation and in protecting autosomes from MSUC.

Importance:Spermatogenic failure (SPGF) is a severe form of male infertility with limited evidence-based medical treatment options. Aromatase inhibitors represent a promising therapeutic strategy for SPGF, but high-quality evidence is lacking. Objective:To evaluate the efficacy and safety of letrozole for improving sperm concentration categories in men with SPGF. Design, Setting, and Participants:This multicenter, open-label, assessor-blinded randomized clinical trial was conducted at 10 male infertility centers in China from July 2023 to March 2024. Men with SPGF (nonobstructive azoospermia [NOA], cryptozoospermia, or severe oligozoospermia) were enrolled. Follow-up for the primary outcome was completed in June 2024. Data were analyzed from July 2024 to March 2025. Interventions:Participants were randomized to receive letrozole (2.5 mg daily) plus vitamins C and E or vitamins C and E alone (control group) for 3 months. Main Outcomes and Measures:The primary outcome was World Health Organization Sperm Concentration Categories (WHO-SCC) upgrade rate at 3 months after randomization. The secondary outcomes were WHO-SCC grades, Dutch Society of Obstetrics and Gynecology Total Motile Sperm Count Categories (NVOG-TMSCC) upgrade rate, semen parameters, and reproductive hormone levels. Results:Among 296 participants (mean [SD] age, 30.2 [3.9] years; 218 [73.6%] with NOA; 147 randomized to the letrozole group and 149 to the control group), 247 (83.4%) completed the trial, and all 296 were included in the primary analysis. WHO-SCC upgrade rates were 14.3% (21 of 147 participants) with letrozole vs 5.4% (8 of 149 participants) with control (risk difference, 9.2% [95% CI, 2.5%-15.8%]; P = .01). Participants in the letrozole group had higher odds of achieving a better WHO-SCC grade than those in the control group (common odds ratio, 2.65 [95% CI, 1.28-5.47]; P = .008). The NVOG-TMSCC upgrade rate was also higher with letrozole. Letrozole significantly increased serum gonadotropins and testosterone while decreasing estradiol. There were no significant between-group differences in semen parameters. Decreased libido (18 [12.2%] vs 8 [5.4%]; P = .04) was more frequent with letrozole than with control. Conclusions and Relevance:In this randomized clinical trial of men with spermatogenic failure, letrozole significantly improved sperm concentration categories and was well tolerated, supporting its use as an option to downstage infertility severity and potentially enable less invasive reproductive management. Trial Registration:chictr.org.cn Identifier: ChiCTR2300073861.

During meiosis, a group of evolutionarily conserved ZMM proteins plays essential roles in stabilizing the recombination intermediates and promoting crossover (CO) formation. In mice, SHOC1 forms a trimeric complex with the other two ZMM proteins, SPO16 and TEX11, to bind recombination intermediates after strand invasion. Although genetic variants of SHOC1 are clinically associated with meiotic arrest and male infertility, their precise molecular mechanisms and evolutionarily conserved functions in human gametogenesis remain enigmatic. Here, we delineated species-specific divergences between human and mouse SHOC1 complex, and identified a missense variant within the XPF-like domain in SHOC1 (c.A1769G:p.Q590R) that was associated with meiotic arrest and non-obstructive azoospermia (NOA). The disorder of the XPF-like domain in SHOC1 impaired DNA double-strand breaks repair by compromising its ability to bind branched DNA structures and the recruitment of M1AP, REDIC1, and ZMM factors to recombination intermediates, ultimately abolishing CO formation. Furthermore, the variant disrupted dynamic 3D chromatin structure in pachytene spermatocytes and induced defects in homologous chromosome synapsis. More importantly, the XPF-like domain in SHOC1 was revealed to prevent autosome intrusion into the sex body compartment, thereby safeguarding critical autosomal loci from meiotic silencing of unsynapsed chromatin (MSUC). Overall, our study demonstrated that the XPF-like domain in SHOC1 is required for homologous recombination and safeguarding autosome from MSUC in meiosis. ### Competing Interest Statement The authors have declared no competing interest. National Key Research and Development Program of China, 2022YFC2702701, 2022YFC2703000 National Natural Science Foundation of China, 82401869, 82371616, 82371607, 82171590, 82401868 Inner Mongolia Academy of Medical Sciences Public Hospital Joint Science and Technology Project, 2023GLLH0045 Scientific Research Startup Funding for High-Level Talents of Taizhou School of Clinical Medicine Fujian Provincial Natural Science Foundation of China, 2023J05271 Scientific Research Startup Funding for High-Level Talents of Taizhou School of Clinical Medicine, TZKY2023RC01

BACKGROUND:Azoospermia, the most severe form of male infertility, is categorized into two types: non-obstructive azoospermia (NOA) and obstructive azoospermia (OA), which exhibit significant genetic heterogeneity. Azoospermia factor (AZF) deletion is a common cause of NOA, whereas congenital bilateral absence of the vas deferens (CBAVD), a severe subtype of OA, is frequently linked to cystic fibrosis transmembrane conductance regulator (CFTR) gene variants. This case report is the first to document the coexistence of a partial AZFa microdeletion and a homozygous CFTR variant in a CBAVD-affected azoospermic patient with intact spermatogenesis. CASE PRESENTATION:A 32-year-old man presented with primary infertility and azoospermia. Clinical evaluation revealed CBAVD (normal hormone levels, low semen volume, pH 6.0, and absence of the vas deferens). Genetic analysis accidentally revealed a 384.9 kb AZFa deletion (sY84 and sY86, but not sY1064, 1182) that removed USP9Y but retained DDX3Y in the proband, his fertile brother, and his father. A homozygous CFTR variant (TG12-5T) was also detected in the proband and his brother and was inherited from heterozygous parental carriers. Microdissection testicular sperm extraction (micro-TESE) revealed intact spermatogenesis, confirmed by histology and immunofluorescence, indicating normal germ cell development. CONCLUSION:This case expands the intricate genetic spectrum of azoospermia by illustrating the critical role of DDX3Y in the AZFa region in spermatogenesis and the variable penetrance of CFTR variant (TG12-5T) in CBAVD. These insights may refine diagnostic strategies and underscore the necessity for tailored fertility management in individuals with multifactorial genetic anomalies.

BACKGROUND:Hemodynamic alterations in the spermatic vein are implicated in infertility among patients with varicocele (VC). Contrast-enhanced ultrasound (CEUS), a powerful tool for hemodynamic analysis, remains unexplored for VC. This study aimed to demonstrate the feasibility of using CEUS to evaluate spermatic vein hemodynamics in patients with VC and establish a clear correlation between specific hemodynamic patterns and impaired semen parameters. This study included 165 patients with left-sided VC and 50 healthy volunteers. All participants underwent CEUS of the spermatic veins, along with maximum venous diameter and testicular volume measurements and serum sex hormone levels and routine semen analyses. The sperm DNA fragmentation index was measured in 146 patients with VC and 37 healthy controls. RESULTS:The analyses revealed four distinct blood flow patterns of the spermatic vein: steady flow, intermittent stasis, intermittent reflux, and filling defect. In healthy spermatic veins, the predominant blood flow patterns included steady flow and intermittent stasis. Spermatic veins with VC exhibited a significant increase in the intermittent reflux and filling defect patterns, with the proportion rising as the clinical grade increased. The four patterns were further grouped into the "steady flow & intermittent stasis" and "intermittent reflux & filling defect" patterns for logistic regression analyses; the intermittent reflux & filling defect pattern was revealed as an independent risk factor for impaired sperm concentration, total sperm counts, progressive motility, morphology, and DNA fragmentation index. CONCLUSIONS:This study validated the feasibility of CEUS for assessing the hemodynamics of the spermatic vein and established the intermittent reflux & filling defect pattern as an independent predictor of impaired semen parameters.

RESEARCH QUESTION:Can a novel hydrogel-encapsulated cryopreservation system reduce cryoinjury while maintaining the viability and functionality of testicular tissue, and support subsequent in-vitro culture for fertility preservation? DESIGN:Various pre-treatment methods that combined cryoprotectants (CPA) with gelatin methacryloyl (GelMA) hydrogels to enhance the rapid freezing of encapsulated testicular tissue were investigated. Key parameters, including CPA concentrations and compositions, were evaluated alongside different cryocarriers to establish an effective freezing system. Fresh and frozen-warmed testicular tissues were cultured in vitro for 1week, comparing the effects of two hydrogel systems: agarose and alginate methacryloyl (AlgMA), based on organ culture method. RESULTS:Encapsulating testicular tissue with GelMA hydrogel, followed by the application of a 30% penetrating CPA, was identified as the optimal pre-treatment strategy. The combination of 15% dimethyl sulfoxide and 15% ethylene glycol significantly enhanced mitochondrial activity compared with 30% concentrations of either CPA alone (both P < 0.05). Among the cryocarriers tested, cryovials demonstrated superior preservation of cellular viability, mitochondrial function and antioxidant capacity. After in-vitro culture, AlgMA hydrogels further preserved basement membrane morphology and germ cell survival in frozen-warmed testicular tissues at levels comparable to fresh tissue, outperforming the traditional agarose hydrogels system in minimizing nuclear condensation, preventing cell loss and preserving tubular structure. CONCLUSIONS:GelMA hydrogel functions as an osmotic barrier, regulating CPA diffusion rates to prevent apoptosis, oxidative stress and other cryoinjuries typically caused by direct exposure to high CPA concentrations. These findings advance the field of fertility preservation, showcasing the potential of novel materials and technologies to improve male fertility restoration outcomes.

Objective For prepubertal and urgently treated malignant tumor patients, ovarian tissue cryopreservation and transplantation represent more appropriate fertility preservation methods. Current clinical practices often involve freezing ovarian tissue with high concentrations of cryoprotectants (CPAs) and thawing with water baths. These processes lead to varying degrees of toxicity and devitrification damage to ovarian tissue. Therefore, this paper proposes optimized methods for vitrification of ovarian tissues based on sodium alginate hydrogel encapsulation and magnetic induction nanowarming technology. Methods Firstly, the study investigated the effects of sodium alginate concentration, the sequence of hydrogel encapsulation and CPAs loading on vitrification efficiency of encapsulated ovarian tissue. Additionally, the capability of sodium alginate hydrogel encapsulation to reduce the required concentration of CPAs was validated. Secondly, a platform combining water bath and magnetic induction nanowarming was established to rewarm ovarian tissue under various concentrations of magnetic nanoparticles and magnetic field strengths. The post-warming follicle survival rate, antioxidant capacity, and ovarian tissue integrity were evaluated to assess the efficacy of the method. Results The study found that ovarian tissue encapsulated with 2% sodium alginate hydrogel exhibited the highest follicle survival rate after vitrification. The method of loading CPAs prior to encapsulation proved more suitable for ovarian tissue cryopreservation, effectively reducing the required concentration of CPAs by 50%. A combination of 8 g/L Fe3O4 nanoparticles and an alternating magnetic field of 300 Gs showed optimal warming effectiveness for ovarian tissue. Combining water bath rewarming with magnetic induction nanowarming yielded the highest follicle survival rate, enhanced antioxidant capacity, and preserved tissue morphology. Conclusion Sodium alginate hydrogel encapsulation of ovarian tissue reduces the concentration of CPAs required during the freezing process. The combination of magnetic induction nanowarming with water bath provides an efficient method ovarian tissue rewarming. This study offers novel approaches to optimize ovarian tissues vitrification.

Non-obstructive azoospermia (NOA) represents the severe form of male infertility, affecting approximately 1% of men during their reproductive years. It is marked by the absence of sperm production caused by testicular dysfunction and has many genetic origins. However, the genetic factors underlying most NOA cases are still unclear. Meiosis, a crucial process ensuring accurate chromosome segregation and generating genetic diversity in gametes, is susceptible to genetic disruptions that may result in NOA. In this study, whole exome sequencing (WES) was conducted on 969 NOA patients, identifying six compound heterozygous KCTD19 variants in three Chinese pedigrees. KCTD19 has been demonstrated to interact with ZFP541 and HDAC1, thereby participating in the modulation of chromatin remodeling and transcriptional programs during meiosis in mice. Herein, our findings expand the phenotypic and mutational spectrum of KCTD19 in male infertility and provide further insights into its role during meiosis. This research underscores the importance of KCTD19 in meiotic progression and male fertility, highlighting the need for further investigation into the molecular mechanisms underlying gametogenic failure in NOA.

The technological advancements in single-cell transcriptome analysis make significant progress in both depth and breadth. However, balancing the cell analysis throughput with full-length transcript coverage remains a persistent challenge. Here, CBTi-seq (Combinational Barcoded Tn5 Transposon Insertion sequencing) is reported, leveraging Tn5 transposase-mediated molecular assembly of combinatorial barcodes and unique molecular identifiers (UMIs) to enable high-resolution multiplexed sequencing of the full-length transcriptome in single cells. This approach achieves molecular resolution by end-to-end sequencing, enabling unambiguous reconstruction of splice variants and structural variations with base-pair precision. The design of orthogonal combination barcode Tn5 reduces DNA barcode diversity while enhancing multiplexing flexibility, and Tn5-delivered UMIs insertion eliminates read bias, providing accurately quantifies transcript abundance through the tagging of each fragment. The method is compatible with both single-cell and spatially resolved tissue microenvironment. Compared with commercial terminal library and other full-length sequencing methods, CBTi-seq achieves superior sensitivity and resolution while significantly reducing costs and work time (≈5 h). Moreover, cell-type-specific alternative splicing patterns are robustly identified in both gene-edited cells and human testicular cells, leveraging this high-resolution capability to further reveal modality dynamic events and isoform switching independent of gene expression changes during spermatogenesis with the potential to reproductive development and diagnostic treatment.

Abstract In mouse early pachytene spermatocytes, the X and Y chromosomes undergo rapid non-homologous (NH) synapsis and desynapsis, but the functional significance remains unknown. Here, we report that pachynema-specific knockout of Speedy A (SpdyA) from telomeres caused persistent Y-X NH synapsis, with the entire Y axis synapsed onto the X axis. This persistent Y-X NH synapsis did not interrupt meiotic sex chromosome inactivation, recombination, or sex body formation, but it disrupted X-Y loop-axis organization and homologous X-Y desynapsis, leading to spermatocyte death. Similarly, persistent Y-X NH synapsis was also observed in pachytene spermatocytes lacking TRF1, where SpdyA was frequently lost from the X-Y non-pseudoautosomal region (non-PAR) telomeres. Mechanistic studies revealed that Serine 48 of SUN1 is a key SpdyA/CDK2 phosphorylation site required for Y-X NH desynapsis. We propose that SpdyA governs Y-X NH desynapsis by stabilizing the linkage between the X-Y non-PAR telomeres and their LINC complexes, and that this process is regulated independently from other aspects of pachynema progression. Our findings suggest a key role for Y-X NH desynapsis in establishing proper X-Y loop-axis organization.

Meiosis is essential for gametogenesis and the maintenance of fertility. Central to this process is meiotic recombination, a mechanism that ensures accurate chromosome segregation and drives genetic diversity. Non-obstructive azoospermia (NOA), a severe form of male infertility, often results from meiotic arrest. Although monogenic variants in genes critical for meiosis have been identified as a cause, the etiology of approximately 60–70