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.

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.

目的:分析不同病因的非梗阻性无精子症(non-obstructive azoospermia,NOA)患者经显微镜下睾丸取精术(microdis-section testicular sperm extraction,micro-TESE)的治疗结局.方法:回顾性分析上海市第一人民医院2015年3月—2022年1月1 355例接受micro-TESE的患者,病因/危险因素包括克氏综合征(Klinefelter syndrome,KS)、Y染色体AZFc缺失、隐睾、腮腺炎性睾丸炎、放化疗、精索静脉曲张以及特发性NOA,研究分析各组患者精子获取率(sperm retrieval rate,SRR),并比较各组取精成功的妊娠结局.结果:NOA患者的总体SRR为26.2％(355/1 355),其中腮腺炎性睾丸炎SRR最高(75.9％,22/29),其次分别为隐睾(70.5％,43/61)、Y染色体AZFc缺失(55.6％,30/54)、KS(47.6％,71/149)、特发性NOA(18.6％,167/897)、放化疗(15.4％,2/13),精索静脉曲张SRR最低(13.2％,20/152).根据手术结局,将NOA患者分为取精成功组及取精失败组.特发性NOA及放化疗类型中,取精成功组卵泡刺激素(follicle-stimulating hormone,FSH)和黄体生成素(luteinizing hormone,LH)水平显著高于取精失败组;Y染色体AZFc缺失类型中,取精成功组FSH、LH水平显著低于取精失败组;腮腺炎性睾丸炎类型中,取精成功组睾丸体积高于取精失败组.回归分析发现年龄可作为预测特发性NOA患者取精结局的因素,年龄较高者拥有较好的取精结局.卵胞浆内单精子注射治疗的妊娠率为51.4％(200/389),活产率为73.5％(147/200).结论:不同病因/危险因素NOA患者的SRR具有显著差异,是影响micro-TESE取精结局的重要指标.

Stepwise mini-incision microdissection testicular sperm extraction (mTESE) is a procedure that attempts to minimize testicular damage. However, the mini-incision approach may vary in patients with different etiologies. Here, we performed a retrospective analysis of 665 men with nonobstructive azoospermia (NOA) who underwent stepwise mini-incision mTESE (Group 1) and 365 men who underwent standard mTESE (Group 2). The results showed that the operation time (mean ± standard deviation) for patients with successful sperm retrieval in Group 1 (64.0 ± 26.6 min) was significantly shorter than that in Group 2 (80.2 ± 31.3 min), with P<0.001. The total sperm retrieval rate (SRR) was 23.1% in our study, and there was no significant difference between Group 1 and Group 2 (P>0.05), even when the etiologies of NOA were taken into consideration. The results of consecutive multivariate logistic regression analysis (odds ratio [OR]: 0.57; 95% confidence interval [CI]: 0.38–0.87; P=0.009) and receiver operating characteristic (ROC) analysis (area under the ROC curve [AUC]=0.628) showed that preoperative anti-Müllerian hormone (AMH) level in idiopathic NOA patients was a potential predictor for surgical outcomes after initial three small incisions made in the equatorial region without sperm examined under an operating microscope (Steps 2–4). In conclusion, stepwise mini-incision mTESE is a useful technique for NOA patients, with comparable SRR, less surgical invasiveness, and shorter operation time compared with the standard approach. Low AMH levels may predict successful sperm retrieval in idiopathic patients even after a failed initial mini-incision procedure.

The regulation of spermatogonial proliferation and apoptosis is of great significance for maintaining spermatogenesis. The single-cell RNA sequencing (scRNA-seq) analysis of the testis was performed to identify genes upregulated in spermatogonia. Using scRNA-seq analysis, we identified the spermatogonia upregulated gene origin recognition complex subunit 6 ( Orc6 ), which is involved in DNA replication and cell cycle regulation; its protein expression in the human and mouse testis was detected by western blot and immunofluorescence. To explore the potential function of Orc6 in spermatogonia, the C18-4 cell line was transfected with control or Orc6 siRNA. Subsequently, 5-ethynyl-2-deoxyuridine (EdU) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, flow cytometry, and western blot were used to evaluate its effects on proliferation and apoptosis. It was revealed that ORC6 could promote proliferation and inhibit apoptosis of C18-4 cells. Bulk RNA sequencing and bioinformatics analysis indicated that Orc6 was involved in the activation of wingless/integrated (Wnt)/β-catenin signaling. Western blot revealed that the expression of β-catenin protein and its phosphorylation (Ser675) were significantly decreased when silencing the expression of ORC6. Our findings indicated that Orc6 was upregulated in spermatogonia, whereby it regulated proliferation and apoptosis by activating Wnt/β-catenin signaling.

Cryopreservation of rare testicular-retrieved spermatozoa for intracytoplasmic sperm injection (ICSI) in patients with severe oligozoospermia and azoospermia remains a major challenge in clinical practice. This study evaluated the Cryopiece system as a potential technique to cryopreserve rare human spermatozoa for ICSI. Small numbers of ejaculated (24 patients) and testicular (13 patients) spermatozoa were cryopreserved using the Cryopiece system. The total number of recovered spermatozoa and motility were assessed after thawing. Thirty-seven couples underwent ICSI using spermatozoa cryopreserved by the Cryopiece system, and ICSI outcomes (rates of fertilization, embryo cleavage, and clinical pregnancy) were evaluated. The average sperm post-thaw retrieval rate was 79.1%, and motility was 29.7%. Ejaculated spermatozoa had a higher post-thaw motility (32.5%) than testicular spermatozoa (21.8%; P = 0.005). ICSI achieved a fertilization rate of 61.9%, embryo cleavage rate of 84.6%, and clinical pregnancy rate of 43.3%. The ICSI outcomes in the ejaculated and testicular frozen-thawed spermatozoa were similar. Assisted oocyte activation (AOA) after ICSI with motile (72.1%) or immotile (71.9%) spermatozoa resulted in a significantly higher fertilization rate than that when using motile spermatozoa without AOA (52.0%; P = 0.005). However, AOA did not enhance the clinical pregnancy rate (55.6% or 40.0% vs 35.3%; P = 0.703). The Cryopiece system is simple and useful for the cryopreservation of small numbers of ejaculated or testicular spermatozoa for ICSI in patients with severe oligozoospermia or nonobstructive azoospermia.

Numerous genes have been associated with multiple morphological abnormalities of the sperm flagella (MMAF), which cause severe asthenozoospermia and lead to male infertility, while the causes of approximately 50% of MMAF cases remain unclear. To reveal the genetic causes of MMAF in an infertile patient, whole-exome sequencing was performed to screen for pathogenic genes, and electron microscope was used to reveal the sperm flagellar ultrastructure. A novel heterozygous missense mutation in the outer dense fiber protein 2 (ODF2) gene was detected, which was inherited from the patient's mother and predicted to be potentially damaging. Transmission electron microscopy revealed that the outer dense fibers were defective in the patient's sperm tail, which was similar to that of the reported heterozygous Odf2 mutation mouse. Immunostaining of ODF2 showed severe ODF2 expression defects in the patient's sperm. Therefore, it was concluded that the heterozygous mutation in ODF2 caused MMAF in this case. To evaluate the possibility of assisted reproductive technology (ART) treatment for this patient, intracytoplasmic sperm injection (ICSI) was performed, with the help of a hypo-osmotic swelling test and laser-assisted immotile sperm selection (LAISS) for available sperm screening, and artificial oocyte activation with ionomycin was applied to improve the fertilization rate. Four ICSI cycles were performed, and live birth was achieved in the LAISS-applied cycle, suggesting that LAISS would be valuable in ART treatment for MMAF.

Spermatogonial stem cells are the foundation of continuous spermatogenesis in adult mammals. Xenograft models have been established to define human SSCs, mostly using infertile and immune-deficient mice as the recipients for human germ cell transplantation. However, it is time-consuming to prepare such recipients using irradiation or chemotherapeutic agents, and this approach may also introduce confounding factors when residual endogenous germ cells recover in transplanted recipients. It remains to be determined whether immune-competent genetically infertile mice can be suitable recipients for xenotransplantation. In this study, we observed similar engraftment efficiencies when using spermatogonia from human biopsied testes across immune-deficient nude mice, immune-competent ICR mice, and genetically infertile Kitw/w-v mice, suggesting minimal immunological rejection from immune-competent mouse recipients upon xenotransplantation of human germ cells. More importantly, we derived EpCAM negative and TNAP positive spermatogonia-like cells (SLCs) from human pluripotent stem cells (PSCs), which highly expressed spermatogonial markers including PLZF, INTERGRINα6, TKTL1, CD90, and DRMT3. We found that upon transplantation, these SLCs proliferated and colonized at the basal membrane of seminiferous tubules in testes of both immune-deficient nude mice and Kitw/w-v mice, though complete spermatogenesis would likely require supporting human signaling factors and microenvironment. Taken together, our study functionally defined the cell identity of PSC-derived SLCs, and supported xenotransplantation using genetically infertile recipients as a convenient model for functionally evaluating spermatogonia derived from different species.

Objective: To analyze the spatio-temporal expression profile of angiotensin-converting enzyme 2 (ACE2), the receptor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in human and mouse testes based on single-cell RNA sequencing (scRNA-Seq). Methods: Ten testicular tissues from humans and nine testicular tissues from C57BL/6 mice with normal developmental stages were collected and digested into single cell suspensions by enzyme, and then the cell-gene expression matrixes were obtained by scRNA-Seq standard processing procedures. After quality control, data standardization, batch effect processing, clustering, and dimensionality reduction, each subgroup of cells was annotated based on known testicular cell bio-markers to clarify the expression patterns and differences of ACE2 in human and mouse testes with normal developmental stages. Results: In this study, nine testicular cell subgroups found in human and mice were identified, including three subgroups of germ cells (spermatogonia, spermatocytes, and spermatids/sperm) and six subgroups of somatic cells (Sertoli cells, macrophages, vascular smooth muscle cells, endothelial cells, Leydig cells, and peritubular myoid cells). In terms of spatial distribution, ACE2 was predominantly expressed in human Sertoli cells in adult testes, and also expressed in Leydig cells, peritubular myoid cells, and germ cells. In terms of time scale, the transcription abundance of ACE2 in human Sertoli cells increased with the development of testis, and the expression level of ACE2 in Sertoli cells after puberty was significantly higher than that in infancy and childhood (P=0.000). Judging from the stages of mouse testicular development, the expression patterns of Ace2 were both significantly different from those in humans. In testis of the 5-week-old adult C57BL/6 mouse, the transcription level of Ace2 was low and it was mainly expressed in vascular smooth muscle cells (P=0.000), while the number of Ace2 positive cell in Sertoli cells was extremely low. Conclusion: SARS-CoV-2 may mainly infects human testis through Sertoli cells, and the conventional C57BL/6 mouse model is not suitable to simulate the effect of SARS-CoV-2 infection on human testicular function.

Background: Cryopreservation of extremely few spermatozoa is still a major challenge for male fertility preservation. This study aims to evaluate the cooling rate, recovery rate, and retrieval rate, along with other parameters of spermatozoa that cryopreserved using Cryopiece, a novel carrier, for individual sperm cryopreservation. Methods: Semen samples from 60 fertile donors were collected, and each semen sample was screened for motile sperm and mixed with cryoprotective agent (CPA), and then frozen using Cryopiece, micro-straw, and mini-straws. The cooling rate, retrieval rate, and recovery rate, morphology, DNA fragmentation index (DFI) and mitochondrial membrane potential (MMP), were compared among the un-frozen sperm and the sperm cryopreserved using these carriers. Results: Cryopiece possessed the fastest cooling rate. After freeze-thaw, the average retrieval rate of sperm cryopreserved using Cryopiece was 96.25%, and the average recovery rate was 64.40%, which were higher than that of sperm cryopreserved using the other two carriers (71.42% and 54.30% for micro-straw, and 63.54% and 58.04% for mini-straw, respectively). There was no significant impact on DFI after sperm cryopreservation, and no significant difference in morphology between sperm cryopreserved using these carriers was observed. Though MMP of sperm changed significantly after cryopreservation, micro-straw maintained sperm MMP better than Cryopiece and mini-straw did, while no significant difference was observed in MMP between sperm cryopreserved using Cryopiece and mini-straw. Conclusions: Cryopiece produced satisfying retrieval and recovery rates in sperm cryopreservation and should be an ideal carrier for cryopreservation of small number of sperm.

Testis-expressed gene 11 (TEX11) mutation has been associated with non-obstructive azoospermia (NOA) and meiotic arrest. An analogous mutation of TEX11 in the mouse impairs meiosis and can be rescued by in vitro expansion of SSCs and gene therapy. However, a lack of genetic screening of a large cohort of Asian patients (including pedigree analysis) and proper functional evaluation limit the clinical application of TEX11 mutation screening. Thus, we performed whole-exome sequencing (WES) in 479 patients with NOA and identified three novel mutations (two splicing mutations and one missense mutation) in TEX11 in three pairs of siblings from three families and four novel pathogenic mutations (three frameshift mutations and a non-sense mutation) of TEX11 in four sporadic NOA-affected cases. Novel variants among family members were segregated by disease phenotype, and all the seven mutations were predicted to be pathogenic. Histological analysis showed that three patients with TEX11 mutations underwent meiotic arrest. The four mutations that resulted in protein truncations and defective meiosis-specific sporulation domain SPO22 were validated by Western blot. In total, we find seven of 479 patients of NOA (1.5%) carrying TEX11 mutations. Our study expands the knowledge of mutations of TEX11 gene in Asian patients with NOA. The high prevalence and X-linked inherited mode indicated that TEX11 might be included in genetic screening panels for the clinical evaluation of patients with NOA.

You have accessJournal of UrologyInfertility: Epidemiology & Evaluation I (MP26)1 Apr 2020MP26-12 RELIABLE SINGLE SPERM CRYOPRESERVATION IN CRYOPIECE FOR AZOOSPERMIA MANAGEMENT Chuan Huang*, Feng Liu, Zi-Jue Zhu, Yan Hone, and Zheng Li Chuan Huang*Chuan Huang* More articles by this author , Feng LiuFeng Liu More articles by this author , Zi-Jue ZhuZi-Jue Zhu More articles by this author , Yan HoneYan Hone More articles by this author , and Zheng LiZheng Li More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000865.012AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Cryopreservation of a single or small number spermatozoa is still a major challenge for male fertility preservation, although previous methods for cryopreservation of a single or small numbers of human spermatozoa have been proposed as a solution for cases of severe male infertility. Many drawbacks have prevented their widespread use, including cumbersome preparation and sperm retrieval procedures, and the fact that the thawed spermatozoa are not immediately available for micromanipulation and required additional treatment which posed excess risk of harm. Our aim was to answer this question: Is a Cryopiece an efficient carrier for freezing a single or small number of human spermatozoa for ICSI? METHODS: we conducted a prospective cohort study of ICSI cycles in men suffering from severe oligospermia or azoospermia, at the Shanghai General Hospital, from 2015 through 2019. In patients with severe oligospermia or azoospermia who underwent ejaculation or surgical sperm retrieval, motile spermatozoa retrieved were subjected to cryopreservation using the Cryopiece RESULTS: The prospective cohort included 27 cases. We used the Cryopiece to vitrify 573 spermatozoa, of which 573 (100%) were motile. The average number of frozen spermatozoa per patient was 21.2 ± 10.3. After thawing, we retrieved 465 spermatozoa, of which 177 were motile, producing a recovery rate of 81.2%. The average number of thawed spermatozoa was 17.2 ± 9.2. The recovery of 177 thawed motile sperm accounted for 38.1% of all frozen motile spermatozoa. The fertilization rate was 56.2%. Of 206 oocyte retrieval procedures, 11 (40.7%) clinical pregnancies were achieved. CONCLUSIONS: The Cryopiece is a simple efficient carrier, optimizing the protocol for freezing a single or small number of spermatozoa. It may allow for the routine use of frozen spermatozoa after ejaculation or surgical for men suffering from azoospermia and thus avoid repeated surgeries. Source of Funding: NA © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e405-e405 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Chuan Huang* More articles by this author Feng Liu More articles by this author Zi-Jue Zhu More articles by this author Yan Hone More articles by this author Zheng Li More articles by this author Expand All Advertisement PDF downloadLoading ...

ABSTRACTNon-obstructive azoospermia (NOA) affects 1% of men. However, the unknowns of NOA pathogenesis and even normal spermatogenic microenvironment establishment severely limit the clinical efficacy of NOA treatment. We profiled > 80,000 human testicular single-cell transcriptomes from 10 healthy donors spanning the range from infant to adult and 7 NOA patients. Sertoli cells, which form the scaffold in the testicular microenvironment, exhibited the most obvious damages in NOA patients. We identified the roadmap of Sertoli cell maturation. Notably, Sertoli cells of patients with congenital causes (Klinefelter syndrome and Y chromosome microdeletions) are mature but with abnormal immune response, while the cells in idiopathic NOA (iNOA) are basically physiologically immature. Furthermore, inhibition of Wnt signaling promotes the maturation of Sertoli cells from iNOA patients, allowing these cells to regain their ability to support germ cell survival. We provide a novel perspective on the development of diagnostic methods and therapeutic targets for NOA.

Clinical efficacy of treatments against non-obstructive azoospermia (NOA), which affects 1% of men, are currently limited by the incomplete understanding of NOA pathogenesis and normal spermatogenic microenvironment. Here, we profile >80,000 human testicular single-cell transcriptomes from 10 healthy donors spanning the range from infant to adult and 7 NOA patients. We show that Sertoli cells, which form the scaffold in the testicular microenvironment, are severely damaged in NOA patients and identify the roadmap of Sertoli cell maturation. Notably, Sertoli cells of patients with congenital causes (Klinefelter syndrome and Y chromosome microdeletions) are mature, but exhibit abnormal immune responses, while the cells in idiopathic NOA (iNOA) are physiologically immature. Furthermore, we find that inhibition of Wnt signaling promotes the maturation of Sertoli cells from iNOA patients, allowing these cells to regain their ability to support germ cell survival. We provide a novel perspective on the development of diagnostic methods and therapeutic targets for NOA.

Objectives This study is designed to generate and propagate human spermatogonial stem cells (SSCs) derived from human pluripotent stem cells (hPSCs). Methods hPSCs were differentiated into SSC-like cells (SSCLCs) by a three-step strategy. The biological characteristics of SSCLCs were detected by immunostaining with antibodies against SSC markers. The ability of self-renewal was measured by propagating for a long time and still maintaining SSCs morphological property. The differentiation potential of SSCLCs was determined by the generation of spermatocytes and haploid cells, which were identified by immunostaining and flow cytometry. The transcriptome analysis of SSCLCs was performed by RNA sequencing. The biological function of SSCLCs was assessed by xeno-transplantation into busulfan-treated mouse testes. Results SSCLCs were efficiently generated by a 3-step strategy. The SSCLCs displayed a grape-like morphology and expressed SSC markers. Moreover, SSCLCs could be propagated for approximately 4 months and still maintained their morphological properties. Furthermore, SSCLCs could differentiate into spermatocytes and haploid cells. In addition, SSCLCs displayed a similar gene expression pattern as human GPR125 + spermatogonia derived from human testicular tissues. And more, SSCLCs could survive and home at the base membrane of seminiferous tubules. Conclusion SSCLCs were successfully derived from hPSCs and propagated for a long time. The SSCLCs resembled their counterpart human GPR125 + spermatogonia, as evidenced by the grape-like morphology, transcriptome, homing, and functional characteristics. Therefore, hPSC-derived SSCLCs may provide a reliable cell source for studying human SSCs biological properties, disease modeling, and drug toxicity screening.

Dear Editor, Sex in mammals is genetically determined and defined at the cellular level by the sex chromosome constitution (XY males and XX females) and at the phenotypic level by the development of gender-specific anatomy, physiology, and behavior.1 46, XY complete gonadal dysgenesis (46, XY CGD), first reported by Swyer in 1955,2 is a rare congenital condition with completely or partially disordered gonadal development, leading to discordance between the genetic, gonadal, and phenotypic sex. Affected patients have a 46, XY karyotype, a female phenotype, normal female external genitalia, and CGD ("streak gonads") without sperm production and follicular or steroid function.3 Moreover, the streak gonads in individuals with 46, XY CGD have a high propensity toward malignancy, and especially dysgerminoma, which occurs in about 20%–50% of patients.1 Most cases of 46, XY CGD are sporadic, but familial cases have also been reported. It is estimated that mutations in sex-determining region Y (SRY) gene were found in 30% of 46, XY CGD cases, and other cases have unknown genetic origins,4 although mutations in several genes involved in testis differentiation may be causative, such as nuclear receptorsubfamily 5 group A member 1 (NR5A1), SRY-related HMG box genes 9 (SOX9), Wilms' tumor-4 (WNT4), dosage-sensitive sex reversal (DAX1), and desert hedgehog (DHH).5 Here, we describe a de novo frameshift mutation leading to a truncated and dysfunctional form of the SRY protein. A 35-year-old female, married for 7 years, was referred to the urology clinic because of concerns about gonadal tumors. She presented for evaluation at 29 years due to the absence of menarche. Physical examination showed a patient 1.64-m tall and weighing 57.5 kg with a female appearance and voice and with no beard or laryngeal prominence. Tanner Stage II breast development and Tanner Stage I pubic hairs were noted. Gynecological examination confirmed normal female external genitalia, and the vagina was well canalized. No palpable mass was identified in the groin. Cytogenetic analysis revealed a karyotype of 46, XY with no evidence of mosaicism, and C-banding analysis further confirmed the existence of a Y chromosome. Y-chromosome microdeletions detected by multiplex polymerase chain reaction (PCR) analysis showed the presence of SRY gene. Pelvis imaging with ultrasonography and computed tomography (CT) scanning revealed a hypoplastic uterus with a thin endometrium and bilateral fallopian tubes, while no ovaries were visualized. Laparoscopy was performed to detect the ovaries and evaluate the risk of tumor development. The bilateral streak gonads were noted (Figure 1a) and then resected. Histological examination of the fallopian tube and streak gonad showed nonneoplastic changes. Secretory cells and ciliated cells could be found in the oviduct (Figure 1b). Primordial follicles were detected in ovarian tissue (Figure 1c).Figure 1: Laparoscopic, histopathological, and genetical examination of the patient with poor pubertal development. (a) Gonad imaging by laparoscopy. (b) Secretory cells and ciliated cells could be found in the oviduct. (c) Primordial follicles could be seen in the streak gonad (marked by arrow). (d) The deletion nucleotide indicated by the arrows was found in the patient, which was not shared by her father, brother or control. (e) According to the family tree, the SRY mutation is de novo. (f) Schematic description of the full-length and truncated SRY proteins. The p.Asn24Ile (N24I) mutation resides in the nNLS domain, and the fsTer60 resides in the HMG box. Scale bars = 100 mm. SG: streak gonad (marked by arrow); FT: fallopian tube; SRY: sex-determining region Y; NLS: nuclear localization; HMG: high mobility group.On admission, she was asked for a familial history to identify possible causes for the disorder. The patient was born at term by normal delivery to nonconsanguineous parents, and her mother denied any use of hormone drugs or exposure to radioactive substances during her pregnancy. The patient has a younger brother and an 8-year-old nephew. There was no other family history of delayed puberty or infertility. Genomic DNA was isolated from the blood leukocytes of the patient, her parents, her sibling, and five fertile control males. The study was approved by the Ethics Committee of Shanghai General Hospital (Shanghai, China). Written informed consent was obtained from each participant. The human SRY gene (reference sequence: NM_003140.2) was amplified by the following two sets of primers, which were designed using Primer 5.0 (PREMIER Biosoft International, Palo Alto, CA, USA): SRY-forward 01: 5'-gaatacattgtcagggtactagggg-3'; SRY-reverse 01: 5'-caggctcacttctggatgtctta-3'; SRY-forward 02: 5'-gggcaagtagtcaacgttactga-3'; and SRY-reverse 02: 5'-ggctcacttctggatgtcttatttctt-3'. The PCR products were resolved on a 2.0% agarose gel, and then purified with gel (Nest, Wuxi, China). The resulting purified DNA was sequenced. A SRY gene mutation was identified between the patient and the reference genome (reference sequence: NM_003140.2), and then screened against the NCBI known mutation database (www.ncbi.nlm.nih.gov/SNP). Direct screening of the SRY coding region revealed a deletion of adenine (A) at nucleotide position 70 (c.70delA) in the patient (Figure 1d). The mutation was not found in her family members or control individuals, which revealed that the SRY mutation occurred de novo (Figure 1e). The mutation (c.70delA, p. Asn24Ile fsTer60; GenBank: AFG33955.1) led to a frameshift and introduced a premature termination codon at position in place of Glu, resulting in a truncated form of the SRY protein (Figure 1f). The mutation was confirmed by bidirectional sequencing. Human bipotential gonads can differentiate into either testes or ovaries, which is depending on the presence or absence of the SRY gene. Expression of SRY gene above a critical threshold at the appropriate time and place drives the expression of the downstream target gene SOX9 and eventually leads to the formation of pre-Sertoli cells. This further orchestrates the formation of functional testes, which determines the development of male primary and secondary sex characteristics. With lack or dysfunction of SRY, the bipotential gonads instead develop as ovaries, and then female primary and secondary sex characteristics occur. The human SRY gene encodes a protein with 204 amino acids that consists of three domains, a central part of 79 amino acids known as a high mobility group (HMG) box and a C-terminal and an N-terminal domain.6 Most mutations occur within the HMG-box as a single amino acid substitution, which can alter DNA binding or prevent nuclear import of the SRY protein.7 Moreover, recent studies of the three-dimensional structure of the human sex-determining region Y (hSRY)-DNA complex in solution show that some residues of the HMG box domain are involved in nuclear localization (NLS) as well as DNA binding.78 The point mutation identified in the patient modified the residues of the N-terminal NLS (nNLS) and removed the C-terminal NLS (cNLS) residues and the HMG box domain, which abolished the function of the HMG box domain as a transcriptional regulator of the SOX9 gene in the sex determination cascade.9 The SRY gene with this frameshift mutation identified in the patient is unable to induce SOX9 expression and the differentiation of pre-Sertoli cells, which is essential for testis cord formation. The abnormal gonads cannot drive the development of Wolffian ducts and repress the development of fallopian tubes in the early stages of embryogenesis. However, the ovary does not develop normally, and only primordial follicles can be detected in the streak gonad. This is consistent with previous reports on the relationship between mutations in the SRY gene and the 46, XY gonadal dysgenesis.1011 We conclude that the SRY gene mutation is a pathogenic factor for the patient. Although the same point mutation resulting in premature termination and a dysfunctional SRY protein has previously been described,10 we are the first to describe it occurring de novo, as opposed to the previous report where it was inherited from a mosaic father.12 Our data also suggest that a mutation hot spot might exist in this region of the SRY gene. AUTHOR CONTRIBUTIONS XBW and YLL identified the case, conducted the genetic studies, and drafted the manuscript. QYZ and JPC carried out the laparoscopy. ZJZ, YZ, and PL participated in the genetic analysis and PL coordinated to draft the manuscript. QL and ZL conceived of the study and reviewed and edited the manuscript. All authors read and approved the final manuscript. COMPETING INTERESTS All authors declared no competing interests. ACKNOWLEDGMENT This work is supported by the National Key Research and Development Program (Grand No. 2017YFC1002003), the National High-tech Research and Development Program (863) of China (Grand No. 2015AA020404), the National Natural Science Foundation of China (Grand No. 81771637, 81571488), and the Frontier Technology Project of Shanghai (Grand No. SHDC12015122)

The proper assessment of male fertility is essential for diagnosing and treating male infertility. Currently, spermiogram and Johnsen testicular biopsy score counts are used to assess male fertility. However, spermiogram is not a suitable option for non-obstructive azoospermia patients, and Johnsen testicular biopsy scores only represent localized and not the overall spermatogenesis. Whole-mount staining was a novel method for evaluating protein expression in the tissue. Thus, we explored its application in human seminiferous tubules. Testicular biopsies from 57 azoospermia patients were categorized as obstructive azoospermia (OA), maturation arrest (MA) and Sertoli-cells only syndrome (SCOS). We performed whole-mount staining of their seminiferous tubules and evaluated the spermatogonial stem cells (SSCs), differentiated spermatogonia (SG), spermatocytes (SPC) and spermatids (SD) with their respective markers (GFRA1, CD117, SYCP3, and PNA) to assess fertility. GFRA1, CD117, SYCP3, and PNA were not expressed in SCOS patients, whereas all of them were detected in OA patients. In MA patients with arrested spermatogenesis at the SPC stage, GFRA1, CD117, and SYCP3, but not PNA were expressed in the seminiferous tubules. In MA patients with arrested spermatogenesis at the spermatogonia stage, only GFRA1 was expressed in the seminiferous tubules. These results were consistent with the Johnsen testicular biopsy score counts except for one patient, where although only Sertoli cells were indicated by the score, SSCs were also detected in the whole-mounts. Collectively, whole-mount staining could be used to analyze the inherent spermatogenesis of seminiferous tubules through staining of germ cells at different stages. It offers a more accurate and promising faster method for assessing male fertility compared with traditional biopsy screening. And it could have potential value for the clinical purpose for male fertility management.

精子发生障碍导致严重精子畸形与无精子症,由此引起的不育症与出生缺陷至少影响500万家庭.目前人类对生精障碍发病机制的认识有限,仅认识到Klinefelter综合征、Y染色体微缺失、隐睾、腮腺炎性睾丸炎影响精子发生.由遗传、环境、生活方式导致精子发生障碍的研究,多集中于啮齿类动物,缺乏对人类精原干细胞增殖分化的研究,尤其对调控信号的分子通路所知不多.人类精子发生障碍研究的重点和难点在于:缺乏基于临床家系的致病基因研究,缺少充足组织样本量支持的应用研究,尚无精子无创评估技术和规范化诊疗体系.本项目针对生精障碍,建立多学科研究团队,指导规范化样本收集,深入开展转化应用研究,建立体内体外向精子分化的研究模型.以此为基础,解析精子形成障碍的遗传学因素,建立精原干细胞向精子定向分化新体系,构建拉曼评估精子与生精小管无创平台并将其用于临床,制订精子发生障碍诊疗路径与诊疗新体系.本项目主要创新和研究成果如下.

Busulfan and other chemotherapeutic drugs used in the treatment of cancer may result in temporary or even permanent damage to spermatogenesis. During spermatogenesis, the rapidly dividing spermatogonia are highly susceptible to chemotherapy. Consequently, there is significant interest in developing an approach that could provide stimulation and regenerate spermatogenesis after chemotherapy. In a previous study, we suggested the potential application for vascular endothelial growth factor C (VEGFC) because of its key role in stimulating the proliferation of spermatogonia. However, methods to facilitate the recovery of spermatogenesis in such patients using VEGFC, or other regulatory factors, are sorely lacking because of the rapid degradation of these proteins and restrictions created by the blood-testis- barrier. To this end, we loaded VEGFC into polyanion dextran sulfate incorporated in a polycation chitosan shell to produce VEGFC sustained-release ultrafine particles (UFPs, CS-DS-VEGFC). We tested such particles in an azoospermic mouse model, created using busulfan. For each mouse, CS-DS-VEGFC was injected into the seminiferous tubules of one testis, while unloaded UFPs (CS-DS), or the VEGFC protein alone, was injected into the opposite testis as a control. All mice were sacrificed and evaluated 5 weeks later. Spermatogenesis in the tubules that were injected with CS-DS-VEGFC was clearly better than those injected with controls, and contained more spermatogonia and spermatocytes, along with Ki67 and PCNA positive- cells per tubule. In addition, the phosphorylation levels of AKT and MAPK in these tubules were also higher than in controls, indicating that CS-DS-VEGFC could induce the sustained activation of these pathways. In conclusion, CS-DS-VEGFC, combined with the efferent tubule injection technique, is a feasible approach with which to improve the regeneration of spermatogenesis in busulfan-induced azoospermic mice.