Rapid azoospermia classification by stimulated Raman scattering and second harmonic generation microscopy.

Disease diagnosis and classification pose significant challenges due to the limited capabilities of traditional methods to obtain molecular information with spatial distribution. Optical imaging techniques, utilizing (auto)fluorescence and nonlinear optical signals, introduce new dimensions for biomarkers exploration that can improve diagnosis and classification. Nevertheless, these signals often cover only a limited number of species, impeding a comprehensive assessment of the tissue microenvironment, which is crucial for effective disease diagnosis and therapy. To address this challenge, we developed a multimodal platform, termed stimulated Raman scattering and second harmonic generation microscopy (SRASH), capable of simultaneously providing both chemical bonds and structural information of tissues. Applying SRASH imaging to azoospermia patient samples, we successfully identified lipids, protein, and collagen contrasts, unveiling molecular and structural signatures for non-obstructive azoospermia. This achievement is facilitated by LiteBlendNet-Dx (LBNet-Dx), our diagnostic algorithm, which achieved an outstanding 100% sample-level accuracy in classifying azoospermia, surpassing conventional imaging modalities. As a label-free technique, SRASH imaging eliminates the requirement for sample pre-treatment, demonstrating great potential for clinical translation and enabling molecular imaging-based diagnosis and therapy.

Identification of ferroptotic genes and phenotypes in idiopathic nonobstructive azoospermia.

Effective treatments for nonobstructive azoospermia (NOA), which affects 1% of all men globally, are limited by undefined pathogenic mechanisms, especially in idiopathic NOA (iNOA). Here, we tried to identify the functional ferroptosis-related genes and phenotypes involved in iNOA. Differentially expressed ferroptotic genes were identified from iNOA mRNA microarray datasets by bioinformatic analyses, and these ferroptotic genes were subsequently filtered by various algorithms. Then, receiver operating characteristic (ROC) curves were generated to evaluate the diagnostic ability of the abovementioned genes for iNOA. Generally, 11 differentially expressed ferroptotic genes were downregulated, and five genes were upregulated in iNOA samples. Four genes, including DUSP1, GPX4, HSD17B11, and SLC2A8, were technically selected and determined to be potential biomarkers for iNOA. Subsequently, similar expression levels were validated at both the RNA and protein levels in the iNOA specimens. Finally, morphologic and biochemical assays were applied to define the ferroptotic phenotypes in testes. The ferroptotic features, like shrunken mitochondria with electron-dense membranes and a reduction in cristae were observed across various cell types within iNOA patients, accompanied by the overload of ferrous ions and increased lipid peroxidation production. Our findings demonstrated that these ferroptosis genes could be involved in the underlying pathogenesis mechanisms of iNOA by regulating ferroptosis and serve as potential diagnostic biomarkers. Also, the ferroptotic phenotypes were identified in iNOA patients.

Single-cell multi-omics sequencing of human spermatogenesis reveals a DNA demethylation event associated with male meiotic recombination.

Human spermatogenesis is a highly ordered process; however, the roles of DNA methylation and chromatin accessibility in this process remain largely unknown. Here by simultaneously investigating the chromatin accessibility, DNA methylome and transcriptome landscapes using the modified single-cell chromatin overall omic-scale landscape sequencing approach, we revealed that the transcriptional changes throughout human spermatogenesis were correlated with chromatin accessibility changes. In particular, we identified a set of transcription factors and cis elements with potential functions. A round of DNA demethylation was uncovered upon meiosis initiation in human spermatogenesis, which was associated with male meiotic recombination and conserved between human and mouse. Aberrant DNA hypermethylation could be detected in leptotene spermatocytes of certain nonobstructive azoospermia patients. Functionally, the intervention of DNA demethylation affected male meiotic recombination and fertility. Our work provides multi-omics landscapes of human spermatogenesis at single-cell resolution and offers insights into the association between DNA demethylation and male meiotic recombination.

Semen parameters' mediation effect on the association between advanced paternal age and IVF clinical outcomes: A 10-year retrospective cohort study.

OBJECTIVE: To explore the mediation between advanced paternal age and the outcomes of in vitro fertilization (IVF) in a female-adjusted cohort. METHODS: The study retrospectively included couples undergoing IVF cycles between 2011 and 2020, and whose female partner was free of medical conditions that would significantly worsen clinical outcomes. Data on patient medical conditions, clinical data, and follow-up information were collected. Causal mediation effect analysis adopting both linear/logistic regression and mixed-effects models was carried out to evaluate the effect of paternal age on the outcomes. RESULTS: 21,959 IVF cycles were included in the study. Semen volume, sperm motility and sperm morphology were significantly associated (P value <0.05) with paternal age. A lower fertilization rate was associated with increased paternal age after adjustment for maternal age (adjusted OR = 0.800; 95 % CI, 0.678, 0.943; P value = 0.008). Mediation analysis revealed that A-level sperm rate and progressive rate respectively mediated 37.0 % and 41.0 % of the association between paternal age and fertilization rate. CONCLUSION: Sperm motility rate, especially A-level sperm rate and rapid progressive rate, mediated the association between advanced paternal age and lower fertilization rate in the cycles.

Machine learning-based clustering to identify the combined effect of the DNA fragmentation index and conventional semen parameters on in vitro fertilization outcomes.

BACKGROUND: Previous studies have demonstrated an association between male sperm quality and assisted reproduction outcomes, focusing on the effects of individual parameters and reaching controversial conclusions. The WHO 6th edition manual highlights a new semen assay, the sperm DNA fragmentation index, for use after routine semen examination. However, the combined effect of the sperm DNA fragmentation index (DFI) and routine semen parameters remains largely unknown. METHODS: We assessed the combined effect of the sperm DFI and conventional semen parameters on single fresh conventional IVF outcomes for infertile couples from January 1, 2017, to December 31, 2020. IVF outcomes were obtained from the cohort database follow-up records of the Clinical Reproductive Medicine Management System of the Third Affiliated Hospital of Guangzhou Medical University. An unsupervised K-means clustering method was applied to classify participants into several coexposure pattern groups. A multivariate logistic regression model was used for statistical analysis. RESULTS: A total of 549 live births among 1258 couples occurred during the follow-up period. A linear exposure-response relationship was observed among the sperm DFI, sperm motility, and IVF outcomes. In multivariable adjustment, increased sperm DFI values and decreased sperm motility and semen concentration levels were associated with reduced odds of favourable IVF outcomes. Four coexposure patterns were generated based on the sperm DFI and the studied semen parameters, as follows: Cluster 1 (low sperm DFI values and high sperm motility and semen concentration levels), Cluster 2 (low sperm DFI values and moderate sperm motility and semen concentration levels), Cluster 3 (low sperm DFI values and low sperm motility and semen concentration levels) and Cluster 4 (high sperm DFI values and low sperm motility and semen concentration levels). Compared with those in Cluster 1, participants in Cluster 3 and Cluster 4 had lower odds of a live birth outcome, with odds ratios (95% confidence intervals [CIs]) of 0.733 (0.537, 0.998) and 0.620 (0.394, 0.967), respectively. CONCLUSIONS: When combined with low sperm DFI values, there was no significant difference between high or moderate sperm concentration and motility levels, and both were associated with favourable IVF outcomes. Low sperm parameter levels, even when DFI values remain low, may still lead to poor IVF outcomes. Participants with high sperm DFI values and low sperm motility and semen concentration levels had the worst outcomes. Our findings offer a novel perspective for exploring the joint effects of sperm DFI and routine semen parameter values.

Prenatal and postnatal exposure to polystyrene microplastics induces testis developmental disorder and affects male fertility in mice.

Polystyrene microplastics (PS-MPs) can threaten human health, especially male fertility. However, most existing studies have focused on the adulthood stage of male reproduction toxicity caused by relatively short-term PS-MP exposure. This study aimed to investigate the toxic effect of PS-MPs on testicular development and reproductive function upon prenatal and postnatal exposure. Pregnant mice and their offspring were exposed to 0, 0.5 mg/L, 5 mg/L, and 50 mg/L PS-MPs through their daily drinking water from gestational day 1 to postnatal day (PND) 35 or PND70. We found that PS-MP exposure induced testis development disorder by PND35 and spermatogenesis dysfunction by PND70. By combining RNA sequencing results and bioinformatics analysis, the hormone-mediated signaling pathway, G1/S transition of the mitotic cell cycle, coregulation of androgen receptor activity, and Hippo signaling pathway were shown to be involved in testis development on PND35. The meiotic cell cycle, regulation of the immune effector process, neutrophil degranulation, and inflammation mediated by chemokine and cytokine signaling pathways were associated with disturbed spermatogenesis on PND70. These findings show that prenatal and postnatal exposure to PS-MPs resulted in testis development disorder and male subfertility, which may be regulated by the Hippo signaling pathway and involve an immune reaction.

SRSF10 is essential for progenitor spermatogonia expansion by regulating alternative splicing.

Alternative splicing expands the transcriptome and proteome complexity and plays essential roles in tissue development and human diseases. However, how alternative splicing regulates spermatogenesis remains largely unknown. Here, using a germ cell-specific knockout mouse model, we demonstrated that the splicing factor Srsf10 is essential for spermatogenesis and male fertility. In the absence of SRSF10, spermatogonial stem cells can be formed, but the expansion of Promyelocytic Leukemia Zinc Finger (PLZF)-positive undifferentiated progenitors was impaired, followed by the failure of spermatogonia differentiation (marked by KIT expression) and meiosis initiation. This was further evidenced by the decreased expression of progenitor cell markers in bulk RNA-seq, and much less progenitor and differentiating spermatogonia in single-cell RNA-seq data. Notably, SRSF10 directly binds thousands of genes in isolated THY+ spermatogonia, and Srsf10 depletion disturbed the alternative splicing of genes that are preferentially associated with germ cell development, cell cycle, and chromosome segregation, including Nasp, Bclaf1, Rif1, Dazl, Kit, Ret, and Sycp1. These data suggest that SRSF10 is critical for the expansion of undifferentiated progenitors by regulating alternative splicing, expanding our understanding of the mechanism underlying spermatogenesis.

Targeting APLN/APJ restores blood-testis barrier and improves spermatogenesis in murine and human diabetic models.

Type 2 diabetes mellitus is one of the most prevalent metabolic diseases presenting with systemic pathologies, including reproductive disorders in male diabetic patients. However, the molecular mechanisms that contributing to spermatogenesis dysfunction in diabetic patients have not yet been fully elucidated. Here, we perform STRT-seq to examine the transcriptome of diabetic patients' testes at single-cell resolution including all major cell types of the testis. Intriguingly, whereas spermatogenesis appears largely preserved, the gene expression profiles of Sertoli cells and the blood-testis barrier (BTB) structure are dramatically impaired. Among these deregulate pathways, the Apelin (APLN) peptide/Apelin-receptor (APJ) axis is hyper-activated in diabetic patients' testes. Mechanistically, APLN is produced locally by Sertoli cells upon high glucose treatment, which subsequently suppress the production of carnitine and repress the expression of cell adhesion genes in Sertoli cells. Together, these effects culminate in BTB structural dysfunction. Finally, using the small molecule APLN receptor antagonist, ML221, we show that blocking APLN/APJ significantly ameliorate the BTB damage and, importantly, improve functional spermatogenesis in diabetic db/db mice. We also translate and validate these findings in cultured human testes. Our findings identify the APLN/APJ axis as a promising therapeutic target to improve reproduction capacity in male diabetic patients.

Human obstructive (postvasectomy) and nonobstructive azoospermia - Insights from scRNA-Seq and transcriptome analysis.

A substantial number of male infertility is caused by azoospermia. However, the underlying etiology and the molecular basis remain largely unknown. Through single-cell (sc)RNA sequencing, we had analyzed testis biopsy samples from two patients with obstructive azoospermia (OA) and nonobstructive azoospermia (NOA). We found only somatic cells in the NOA samples and explored the transcriptional changes in Sertoli cells in response to a loss of interactions with germ cells. Moreover, we observed a germ cell population discrepancy between an OA (postvasectomy) patient and a healthy individual. We confirmed this observation in a secondary study with two datasets at GSM3526588 and GSE124263 for detailed analysis wherein the regulatory mechanisms at the transcriptional level were identified. These findings thus provide valuable information on human spermatogenesis, and we also identified insightful information for further research on reproduction-related diseases.

Clinical Outcomes and Live Birth Rate Resulted From Microdissection Testicular Sperm Extraction With ICSI-IVF in Non-Obstructive Azoospermia: A Single-Center Cohort Study.

Background: Most of data available in the literature reported the sperm retrieval rate and limited intracytoplasmic sperm injection (ICSI) results of microdissection testicular sperm extraction (micro-TESE) in non-obstructive azoospermia (NOA) patients with different etiologies. Unfortunately, there is currently a lack of comprehensive data to guide clinicians in conducting comprehensive consultations with NOA patients. Objectives: To obtain more comprehensive evidence-based data and clinical outcomes for better consultation of NOA patients who opted to undergo micro-TESE combined with ICSI-IVF. Methods: It was a retrospective study involved 968 NOA patients underwent micro-TESE during January 2015 to December 2019. Embryological, clinical, and live birth outcomes were demonstrated comprehensively and three kinds of stratification analyses were performed based on ICSI-IVF cycles using frozen and fresh sperm, different etiologies of NOA and various amounts of sperm retrieved. Results: The sperm retrieval rate was 44.6%, and ICSI was performed in 299 couples leading to 150 clinical pregnancies and 140 live-birth deliveries. The clinical pregnancy rate (CPR) was 50.17%, and the cumulative live birth rate (LBR) was 46.82%, and the low birth defects rate was 1.43%. No significant difference was observed about cumulative LBR in frozen sperm group and fresh sperm group (47.5% vs 42.9%, P>0.05). NOA patients with AZFc microdeletions had the lowest rate of a high-score embryo on day 3 (4.4%, P<0.05) and the lowest cumulative LBR (19.4%, P<0.05). NOA patients with lower sperm count (having fewer than 20 sperms retrieved) had significantly lower cumulative LBR than those with higher sperm count (having more than 20 sperms retrieved) (28.1% vs 51.9%, P<0.05). Conclusions: For those NOA patients who stepped in ICSI-IVF cycles, the cumulative LBR was 46.82%. No significant difference was indicated in the LBR between ICSI-IVF cycles using frozen or fresh testicular sperm. Compared to other etiologies, NOA caused by AZFc microdeletions have the poorest embryological and clinical outcomes. Patients with less testicular sperm retrieved have poorer embryological and clinical outcomes.

Gonadotropins treatment prior to microdissection testicular sperm extraction in non-obstructive azoospermia: a single-center cohort study.

BACKGROUND: Microdissection testicular sperm extraction (micro-TESE) in combination with ICSI can make paternity possible for non-obstructive azoospermia (NOA) patients. Testicular sperm can be successfully retrieved in nearly half of NOA patients. Nevertheless, not many convincing protocols are established to improve sperm retrieval rate (SRR). The goal of this study was to evaluate whether gonadotropins therapy before micro-TESE could improve sperm retrieval rate and affect the ICSI outcomes in non-obstructive azoospermia patients with hypergonadotropic hypogonadism. METHODS: This retrospective cohort study included a total of 569 non-obstructive azoospermia men who underwent micro-TESE with or without 3-month of preoperative hCG / hCG plus highly purified urinary FSH (uFSH) between January 2016 and December 2019. The primary outcome was the sperm retrieval rate of micro-TESE. RESULTS: Sperm was found in 27 patients among 395 NOA men who accepted preoperative gonadotropins treatment (6.8%, 27/395) in post-treatment semen analysis for ICSI. One hundred forty nine out of 542 patients could successfully obtain enough sperm for ICSI through the micro-TESE (overall SRR = 27.5%). There was a statistically significant difference in the SRR between the preoperative gonadotropins treatment and non-gonadotropins treatment groups (31.2%, 115/368 vs. 19.5%, 34/174, P = 0.006). In the multivariable analysis with IPTW according to the propensity score, there was a significant association between preoperative gonadotropins treatment and the SRR (OR, 1.59; 95% CI: 1.02-2.52; P = 0.042). No differences in the clinical pregnancy rate, live birth delivery rate, or miscarriage rate were observed between the two groups. CONCLUSION: Preoperative gonadotropins therapy seems to have a role in improving SRR in NOA patients with hypergonadotropic hypogonadism. We found that gonadotropins therapy had no effect on ICSI clinical outcomes and live birth.

A high-throughput, open-space and reusable microfluidic chip for combinational drug screening on tumor spheroids.

Screening drug combinations using tumor spheroids can play a vital role in the development of disease treatment and personalized medicine. However, current studies focus on drug gradients or combinations of two drugs in most cases, and it is difficult to find complex therapeutic combinations involving more drugs. The use of design-of-experiment (DOE) microfluidics is a potential strategy to study this area systematically. Here we develop a high-throughput, open-space multilayered PMMA microfluidic chip for combinational drug screening on tumor spheroids. This microchip is straightforward to fabricate, compatible with standard spheroid cultures, and friendly for end-users. The device consists of an inlet layer and multiple dispersing layers. In the inlet layer, different samples can be loaded into the chip simultaneously. The sample solutions flow into the dispersing layers to generate various combinations based on the specific DOE principle. We demonstrated that the chip performance is in quantitative agreement with the design, using water and doxycycline combinations as models. As a proof-of-concept study, we constructed a HeLa reporter cell line to quantify the autophagy of tumor spheroids and used the chip to identify critical factors relating to the growth of the spheroids. Specifically, we used L-glutamine, D-glucose, FBS, and cisplatin as the factors and studied the autophagy, growth curves, and spheroid sizes in response to different combinations of the four factors. We found that D-glucose can inhibit the effects of cisplatin on tumor spheroids, and cisplatin caused severe autophagy in 3D tumor spheroids compared to 2D monoculture cells. Our method has the potential to allow more drug combinations to be examined, and it can be extended to DOE approaches with seven or more inputs.

[Research progress on screening of pregnancy-related diseases based on fetal cell free DNA].

Fetal cell free DNA (cfDNA) in maternal blood circulation mainly originates from placental trophoblasts which have dual characteristics of apoptotic cells and the embryo, and can be affected by maternal factors. Pregnancy-related diseases including preeclampsia, gestational diabetes mellitus, preeclampsia, macrosomia and fetal growth restriction can seriously affect maternal health and pregnancy outcome. Early prediction and timely intervention are important means to reduce the risk. Fetal cfDNA and prediction of pregnancy-related diseases have become a hot topicfor current research. This paper reviews the latest progress made in the field.

MicroRNA-126 engineered muscle-derived stem cells attenuates cavernosa injury-induced erectile dysfunction in rats.

BACKGROUND: Cavernosa injury is a common cause of organic erectile dysfunction (ED), which requires safe and effective treatments. In the present study, the therapeutic efficiency of muscle-derived stem cells (MDSCs) modified with microRNA-126 (miR-126) was determined in rats with cavernosa injury. METHODS: MDSCs were transfected with miR-126 and then were transplanted into rats with cavernosa injury. Erectile function, vascular function (western blot and immunofluorescence), extraction, and detection of exosomes were then undertaken. RESULTS: On the 28th day after transplantation, the highest value of intra-cavernous pressure (ICP)/mean arterial pressure (MAP) in rats of miRNA-126 group (0.84 ± 0.14) was observed (Control: 0.38 ± 0.07; MDSC: 0.54 ± 0.11, Vector: 0.60 ± 0.02; respectively). Treatment of miRNA-126-modified-MDSCs remarkably strengthened vascular structure, supported by hematoxylin-eosin staining. The expression of CD31, von Willebrand Factor and vascular endothelial factors were higher than those in other groups, indicating improved vascular function. In vitro mechanism studies showed that exosomes containing miR-126 isolated from MDSCs promoted angiogenesis and attenuated apoptosis of human umbilical venous endothelial cells. Finally, insulin receptor substrate 1 and Krüppel-like factor 10 were determined as the direct target genes of miR-126. CONCLUSIONS: MiR-126 engineered MDSCs notably repaired cavernosa injury in rats via vascular reconstruction by directly targeting IRS1 and KLF10, in which the exosomes secreted by MDSCs played a critical role.

Deciphering the autophagy regulatory network via single-cell transcriptome analysis reveals a requirement for autophagy homeostasis in spermatogenesis.

Background: Autophagy has been implicated as a crucial component in spermatogenesis, and autophagy dysfunction can lead to reproductive disorders in animal models, including yeast, C. elegans and mice. However, the sophisticated transcriptional networks of autophagic genes throughout human spermatogenesis and their biological significance remain largely uncharacterized. Methods: We profiled the transcriptional signatures of autophagy-related genes during human spermatogenesis by assessing specimens from nine fertile controls (including two normal persons and seven obstructive azoospermia (OA) patients) and one nonobstructive azoospermia (NOA) patient using single-cell RNA sequencing (scRNA-seq) analysis. Dysregulation of autophagy was confirmed in two additional NOA patients by immunofluorescence staining. Gene knockdown was used to identify the role of Cst3 in autophagy during spermatogenesis. Results: Our data uncovered a unique, global stage-specific enrichment of autophagy-related genes. Human-mouse comparison analysis revealed that the stage-specific expression pattern of autophagy-related genes was highly conserved in mammals. More importantly, dysregulation of some clusters of autophagy-related genes was observed in NOA patients, suggesting the association of autophagy with male infertility. Cst3, a human-mouse conserved and autophagy-related gene that is actively expressed in spermatogonia and early spermatocytes, was found to regulate spermatogonial stem cell (SSC) maintenance and subsequent male germ cell development. Knockdown of Cst3 increased autophagic activity in mouse SSCs and subsequently suppressed the transcription of SSC core factors such as Oct4, Id1, and Nanos3, which could be efficiently rescued by manipulating autophagic activity. Conclusions: Our study provides comprehensive insights into the global transcriptional signatures of autophagy-related genes and confirms the importance of autophagy homeostasis in SSC maintenance and normal spermatogenesis, opening new avenues for further dissecting the significance of the autophagy regulatory network in spermatogenesis as well as male infertility.

Role of treatment with human chorionic gonadotropin and clinical parameters on testicular sperm recovery with microdissection testicular sperm extraction and intracytoplasmic sperm injection outcomes in 184 Klinefelter syndrome patients.

OBJECTIVE: To investigate whether preoperative human chorionic gonadotropin (hCG) treatment can help predict the outcomes of microdissection testicular sperm extraction (micro-TESE) and affect fertility outcomes in non-mosaic Klinefelter syndrome (KS) patients. DESIGN: Retrospective cohort study. SETTING: University-affiliated fertility center. PATIENT(S): A total of 184 non-mosaic KS patients who underwent micro-TESE with or without preoperative hCG treatment from January 2016 to July 2019. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sperm retrieval rate (SRR) with and without hCG treatment, logistic models analysis. RESULT(S): Eighty KS patients (43.5%) had successful sperm retrievals after micro-TESE. There was no statistically significant difference in the SRR between the group who received hCG treatment and the group that did not (44.0% vs. 43.3%). Logistic regression analyses demonstrated that the hCG treatment had no statistically significant effect on successful sperm retrieval. However, higher preoperative testosterone (T) levels seemed to be associated with a higher probability of successful sperm retrieval (multivariate adjusted odds ratio 1.09; 95% confidence interval [CI], 1.04-1.16). The prediction model for SRR on KS patients had an area under the curve of 67.3% (95% CI, 59.3-75.3%). In the hCG treatment group, the data indicated that the three parameters of testicular volume, pretreatment T level, and alterations of T were associated with the probability of successful sperm retrieval. Moreover, hCG therapy did not affect intracytoplasmic sperm injection (ICSI) outcomes. No differences in the pregnancy rate or live-birth rate were observed between the two groups. CONCLUSION(S): Therapy with hCG does not affect SRR or ICSI outcomes of non-mosaic KS patients. However, preoperative T levels, whether treated with hCG or not, can predict the chance of sperm retrieval with micro-TESE.

Adenylate kinase 1 deficiency disrupts mouse sperm motility under conditions of energy stress†.

Mammalian spermatozoa are highly polarized cells characterized by compartmentalized cellular structures and energy metabolism. Adenylate kinase (AK), which interconverts two ADP molecules into stoichiometric amounts of ATP and AMP, plays a critical role in buffering adenine nucleotides throughout the tail to support flagellar motility. Yet the role of the major AK isoform, AK1, is still not well characterized. Here, by using a proteomic analysis of testis biopsy samples, we found that AK1 levels were significantly decreased in nonobstructive azoospermia patients. This result was further verified by immunohistochemical staining of AK1 on a tissue microarray. AK1 was found to be expressed in post-meiotic round and elongated spermatids in mouse testis and subsequent mature sperm in the epididymis. We then generated Ak1 knockout mice, which showed that AK1 deficiency did not induce any defects in testis development, spermatogenesis, or sperm morphology and motility under physiological conditions. We further investigated detergent-modeled epididymal sperm and included individual or mixed adenine nucleotides to mimic energy stress. When only ADP was available, Ak1 disruption largely compromised sperm motility, manifested as a smaller beating amplitude and higher beating frequency, which resulted in less effective forward swimming. The energy restriction/recover experiments with intact sperm further addressed this finding. Besides, decreased AK activity was observed in sperm of a male fertility disorder mouse model induced by cadmium chloride. These results cumulatively demonstrate that AK1 was dispensable for testis development, spermatogenesis, or sperm motility under physiological conditions, but was required for sperm to maintain a constant adenylate energy charge to support sperm motility under conditions of energy stress.

Functional reconstruction of injured corpus cavernosa using 3D-printed hydrogel scaffolds seeded with HIF-1α-expressing stem cells.

Injury of corpus cavernosa results in erectile dysfunction, but its treatment has been very difficult. Here we construct heparin-coated 3D-printed hydrogel scaffolds seeded with hypoxia inducible factor-1α (HIF-1α)-mutated muscle-derived stem cells (MDSCs) to develop bioengineered vascularized corpora. HIF-1α-mutated MDSCs significantly secrete various angiogenic factors in MDSCs regardless of hypoxia or normoxia. The biodegradable scaffolds, along with MDSCs, are implanted into corpus cavernosa defects in a rabbit model to show good histocompatibility with no immunological rejection, support vascularized tissue ingrowth, and promote neovascularisation to repair the defects. Evaluation of morphology, intracavernosal pressure, elasticity and shrinkage of repaired cavernous tissue prove that the bioengineered corpora scaffolds repair the defects and recover penile erectile and ejaculation function successfully. The function recovery restores the reproductive capability of the injured male rabbits. Our work demonstrates that the 3D-printed hydrogels with angiogenic cells hold great promise for penile reconstruction to restore reproductive capability of males.

Molecular recognition-directed site-specific release of stem cell differentiation inducers for enhanced joint repair.

The remarkable difference in cell type and matrix composition between two connected parts of a joint (cartilage and subchondral bone) makes it challenging to simultaneously regenerate both parts for joint repair. Thus we chemically designed a biphasic hydrogel made of two well-bonded shape-tunable hydrogel phases, termed bone-regenerating hydrogel (BRH) and cartilage-regenerating hydrogel (CRH). The BRH and CRH, encapsulating stem cells, were produced by photo-crosslinking bone and cartilage matrix-mimicking biopolymers and a nanobox (ß-cyclodextrin) in situ in the subchondral bone defect and cartilage defect, respectively. The nanoboxes in BRH and CRH were loaded with osteogenic and chondrogenic differentiation inducers (melatonin and kartogenin) by host-guest interactions, respectively. Such interactions directed the sustained phase- and defect site-specific release of the inducers and subsequent site-specific stem cell differentiation into cartilage and bone forming cells for joint repair. The strategy opens up a new chemical approach to biomaterials with phase-specific drug release for tissue repair.