BACKGROUND Spermatogonial stem cells(SSCs)are the origin of male spermatogenesis,which can reconstruct germ cell lineage in mice.However,the application of SSCs for male fertility restoration is hindered due to the un...BACKGROUND Spermatogonial stem cells(SSCs)are the origin of male spermatogenesis,which can reconstruct germ cell lineage in mice.However,the application of SSCs for male fertility restoration is hindered due to the unclear mechanisms of proliferation and self-renewal in humans.AIM To investigate the role and mechanism of SPOC domain-containing protein 1(SPOCD1)in human SSC proliferation.METHODS We analyzed publicly available human testis single-cell RNA sequencing(RNAseq)data and found that SPOCD1 is predominantly expressed in SSCs in the early developmental stages.Small interfering RNA was applied to suppress SPOCD1 expression to detect the impacts of SPOCD1 inhibition on SSC proliferation and apoptosis.Subsequently,we explored the target genes of SPOCD1 using RNA-seq and confirmed their role by restoring the expression of the target genes.In addition,we examined SPOCD1 expression in some non-obstructive azoospermia(NOA)patients to explore the correlation between SPOCD1 and NOA.RESULTS The uniform manifold approximation and projection clustering and pseudotime analysis showed that SPOCD1 was highly expressed in the early stages of SSC,and immunohistological results showed that SPOCD1 was mainly localized in glial cell line-derived neurotrophic factor family receptor alpha-1 positive SSCs.SPOCD1 knockdown significantly inhibited cell proliferation and promoted apoptosis.RNA-seq results showed that SPOCD1 knockdown significantly downregulated genes such as adenylate kinase 4(AK4).Overexpression of AK4 in SPOCD1 knockdown cells partially reversed the phenotypic changes,indicating that AK4 is a functional target gene of SPOCD1.In addition,we found a significant downregulation of SPOCD1 expression in some NOA patients,suggesting that the downregulation of SPOCD1 may be relevant for NOA.CONCLUSION Our study broadens the understanding of human SSC fate determination and may offer new theories on the etiology of male infertility.展开更多
Congenital bilateral absence of the vas deferens(CBAVD)is observed in 1%–2%of males presenting with infertility and is clearly associated with cystic fibrosis transmembrane conductance regulator(CFTR)mutations.CFTR i...Congenital bilateral absence of the vas deferens(CBAVD)is observed in 1%–2%of males presenting with infertility and is clearly associated with cystic fibrosis transmembrane conductance regulator(CFTR)mutations.CFTR is one of the most well-known genes related to male fertility.The frequency of CFTR mutations or impaired CFTR expression is increased in men with nonobstructive azoospermia(NOA).CFTR mutations are highly polymorphic and have established ethnic specificity.Compared with F508Del in Caucasians,the p.G970D mutation is reported to be the most frequent CFTR mutation in Chinese patients with cystic fibrosis.However,whether p.G970D participates in male infertility remains unknown.Herein,a loss-of-function CFTR p.G970D missense mutation was identified in a patient with CBAVD and NOA.Subsequent retrospective analysis of 122 Chinese patients with CBAVD showed that the mutation is a common pathogenic mutation(4.1%,5/122),excluding polymorphic sites.Furthermore,we generated model cell lines derived from mouse testes harboring the homozygous Cftr p.G965D mutation equivalent to the CFTR variant in patients.The Cftr p.G965D mutation may be lethal in spermatogonial stem cells and spermatogonia and affect the proliferation of spermatocytes and Sertoli cells.In spermatocyte GC-2(spd)ts(GC2)Cftr p.G965D cells,RNA splicing variants were detected and CFTR expression decreased,which may contribute to the phenotypes associated with impaired spermatogenesis.Thus,this study indicated that the CFTR p.G970D missense mutation might be a pathogenic mutation for CBAVD in Chinese males and associated with impaired spermatogenesis by affecting the proliferation of germ cells.展开更多
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 eva...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.展开更多
Male infertility is a major reproductive disorder,which is clinically characterized by highly heterogeneous phenotypes of abnormal sperm count or quality.To date,five male patients with biallelic loss-of-function(LOF)...Male infertility is a major reproductive disorder,which is clinically characterized by highly heterogeneous phenotypes of abnormal sperm count or quality.To date,five male patients with biallelic loss-of-function(LOF)variants of PARN-like ribonuclease domain-containing exonuclease 1(PNLDC1)have been reported to experience infertility with nonobstructive azoospermia.The aim of this study was to identify the genetic cause of male infertility with oligo-astheno-teratozoospermia(OAT)in a patient from a Chinese Han family.Whole-exome and Sanger sequencing analyses identified a homozygous LOF variant(NM_173516.2,c.l42C>T,p.Gln48Ter)in PNLDC1.Hematoxylin and eosin staining revealed that the spermatozoa of the patient with OAT had an irregular head phenotype,including microcephaly,head tapering,and globozoospermia.Consistently,peanut agglutinin staining of the spermatozoa revealed a complete or partial loss of the acrosome.Furthermore,the disomy rate of chromosomes in the patient’s spermatozoa was significantly increased compared with that of a fertile control sample.We reported an LOF variant of the PNLDC1 gene responsible for OAT.展开更多
Primary ciliary dyskinesia(PCD)is a congenital,motile ciliopathy with pleiotropic symptoms.Although nearly 50 causative genes have been identified,they only account for approximately 70%of definitive PCD cases.Dynein ...Primary ciliary dyskinesia(PCD)is a congenital,motile ciliopathy with pleiotropic symptoms.Although nearly 50 causative genes have been identified,they only account for approximately 70%of definitive PCD cases.Dynein axonemal heavy chain 10(DNAH10)encodes a subunit of the inner arm dynein heavy chain in motile cilia and sperm flagella.Based on the common axoneme structure of motile cilia and sperm flagella,DNAH10 variants are likely to cause PCD.Using exome sequencing,we identified a novel DNAH10 homozygous variant(c.589C>T,p.R197W)in a patient with PCD from a consanguineous family.The patient manifested sinusitis,bronchiectasis,situs inversus,and asthenoteratozoospermia.Immunostaining analysis showed the absence of DNAH10 and DNALI1 in the respiratory cilia,and transmission electron microscopy revealed strikingly disordered axoneme 9+2 architecture and inner dynein arm defects in the respiratory cilia and sperm flagella.Subsequently,animal models of Dnah10-knockin mice harboring missense variants and Dnah10-knockout mice recapitulated the phenotypes of PCD,including chronic respiratory infection,male infertility,and hydrocephalus.To the best of our knowledge,this study is the first to report DNAH10 deficiency related to PCD in human and mouse models,which suggests that DNAH10 recessive mutation is causative of PCD.展开更多
Dear Editor,Male subfertility,a multifactorial disease affecting~7%of the global male population,is usually caused by abnormalities in sperm flagella.The flagella and motile cilia have similar“9+2”axonemes and are e...Dear Editor,Male subfertility,a multifactorial disease affecting~7%of the global male population,is usually caused by abnormalities in sperm flagella.The flagella and motile cilia have similar“9+2”axonemes and are evolutionarily conserved,being widely distributed in bacteria,archaea and eukaryotes1.Cilia defects also lead to primary ciliary dyskinesia(PCD),which affects approximately 1/10,000 individuals worldwide.展开更多
Sperm morphology was once believed as one of the most predictive indicators of pregnancy outcome in assisted reproductive technology(ART).However,the impact of teratozoospermia on in vitro fertilization(IVF)/intracyto...Sperm morphology was once believed as one of the most predictive indicators of pregnancy outcome in assisted reproductive technology(ART).However,the impact of teratozoospermia on in vitro fertilization(IVF)/intracytoplasmic sperm injection(ICSI)outcomes and its offspring remains inconclusive.In order to evaluate the influence of teratozoospermia on pregnancy outcome and newborn status after IVF and ICSI,a retrospective study was conducted.This was a matched case-control study that included 2202 IVF cycles and 2574 ICSI cycles and was conducted at the Reproductive and Genetic Hospital of CITIC-Xiangya in Changsha,China,from June 2013 to June 2018.Patients were divided into two groups based on sperm morphology:teratozoospermia and normal sperm group.The pregnancy outcome and newborn outcome were analyzed.The results indicated that couples with teratozoospermia had a significantly lower optimal embryo rate compared to those with normal sperm morphology in IVF(P=0.007),while there were no statistically significant differences between the two groups in terms of the fertilization rate,cleavage rate,implantation rate,and pregnancy rate(all P>0.05).Additionally,teratozoospermia was associated with lower infant birth weight in multiple births after IVF.With regard to ICSI,there was no significant difference in both pregnancy outcome and newborn outcome between the teratozoospermia and normal groups(both P>0.05).Furthermore,no increase in the risk of birth defects occurred in the teratozoospermia group after IVF/ICSI.Consequently,we believe that teratozoospermia has limited predictive value for pregnancy outcomes in IVF/ICSI,and has little impact on the resulting offspring if multiple pregnancy is avoided.展开更多
Spermiogenesis is a complex and tightly regulated process,consisting of acrosomal biogenesis,condensation of chromatin,flagellar assembly,and disposal of extra cytoplasm.Previous studies have reported that sperm flage...Spermiogenesis is a complex and tightly regulated process,consisting of acrosomal biogenesis,condensation of chromatin,flagellar assembly,and disposal of extra cytoplasm.Previous studies have reported that sperm flagellar 2(SPEF2)deficiency causes severe asthenoteratozoospermia owing to spermiogenesis failure,but the underlying molecular mechanism in humans remains unclear.Here,we performed proteomic analysis on spermatozoa from three SPEF2 mutant patients to study the functional role of SPEF2 during sperm tail development.A total of 1262 differentially expressed proteins were detected,including 486 upregulated and 776 downregulated.The constructed heat map of the differentially expressed proteins showed similar trends.Among these,the expression of proteins related to flagellar assembly,including SPEF2,sperm associated antigen 6(SPAG6),dynein light chain tctex-type 1(DYNLT1),radial spoke head component 1(RSPH1),translocase of outer mitochondrial membrane 20(TOM20),EF-hand domain containing 1(EFHC1),meiosis-specific nuclear structural 1(MNS1)and intraflagellar transport 20(IFT20),was verified by western blot.Functional clustering analysis indicated that these differentially expressed proteins were specifically enriched for terms such as spermatid development and flagellar assembly.Furthermore,we showed that SPEF2 interacts with radial spoke head component 9(RSPH9)and IFT20 in vitro,which are well-studied components of radial spokes or intra-flagellar transport and are essential for flagellar assembly.These results provide a rich resource for further investigation into the molecular mechanism underlying the role that SPEF2 plays in sperm tail development and could provide a theoretical basis for gene therapy in SPEF2 mutant patients in the future.展开更多
Diminished ovarian reserve(DOR)is a disorder of ovarian function in which the ovary loses its normal reproductive potential,including decreasing oocyte quantity and quality.The disorder is associated with infertility ...Diminished ovarian reserve(DOR)is a disorder of ovarian function in which the ovary loses its normal reproductive potential,including decreasing oocyte quantity and quality.The disorder is associated with infertility in women of reproductive age with regular menses and poor response to controlled ovarian hyperstimulation(Broekmans et al.,2009).展开更多
The clinical applications of acrosin activity are limited.We analyzed 61578 male partners in infertile couples who visited the outpatient department of the Reproductive and Genetic Hospital of CITIC-Xiangya(Changsha,C...The clinical applications of acrosin activity are limited.We analyzed 61578 male partners in infertile couples who visited the outpatient department of the Reproductive and Genetic Hospital of CITIC-Xiangya(Changsha,China)between August 2014 and December 2019 to determine the reference ranges and thresholds for acrosin activity in infertile Chinese men;to determine whether correlations exist between acrosin activity and age,sperm concentration,sperm morphology,or sperm motility;and to evaluate whether acrosin activity could serve as an effective prognostic indicator for choosing between in vitro fertilization(IvF)and intracytoplasmic sperm injection(icsl)in the clinic.The cut-off value for the normal reference range of acrosin activity for male partners in infertile couples was 24.78μlU per 106 sperm.There was no significant association between acrosin activity and age,sperm concentration,semen volume,total sperm count,progressive motility,or total motile spermatozoa.A weak positive correlation was found between acrosin activity and normal sperm morphology.There was a statistically significant difference in abnormal acrosome morphology between the group with high acrosin activity(>24.78μlU per 106 sperm)and the group with low acrosin activity(<24.78μlU per 106 sperm).The group with a low IVF fertilization rate had a high index of abnormal acrosomal morphology at 21.2%,while the group with a high IVF fertilization rate had a low index of 0.2%.At an acrosin activity of<24.78μlU per 10 sperm,in one cycle of the same patient,the fertilization rate,normal fertilization rate,and good-quality embryo rate for Icsl were significantly higher than those for IVF.Therefore,the most promising application of acrosin activity could be in the selection of IcsI over IVF for infertile male patients with complete fertilization failure or a low fertilization rate.展开更多
Objective:This study is to investigate the effect of different single-nucleotide polymorphisms of follicle-stimulating hormone receptor(FSHR)gene on gonadotropin(Gn)administration dosage during controlled ovarian hype...Objective:This study is to investigate the effect of different single-nucleotide polymorphisms of follicle-stimulating hormone receptor(FSHR)gene on gonadotropin(Gn)administration dosage during controlled ovarian hyperstimulation(COH)protocol of in vitro fertilization and embryo transfer.Methods:This retrospective study included 184 Chinese infertile women in Center for Reproduction and Genetics of Suzhou Municipal Hospital from June 2012 to 2014.All of the enrolled patients were homogeneous in some basal characteristics,and they all met the eligibility criteria.Blood tests were conducted on day 3 of menstrual cycle or the day of human chorionic gonadotropin administration for hormonal profile analysis and DNA extraction.DNA sequencing was performed for polymorphism analysis.The participants were classified into threonine(Thr)/Thr,Thr/alanine(Ala),and Ala/Ala groups according to genotype at position 307,and asparagine/asparagine(Asn/Asn),Asn/serine(Ser),and Ser/Ser groups according to genotype at position 680.Logistic regression and correlation analyses were performed to identify the effect of these two polymorphisms on Gn consumption.Results:The frequency of Thr307Ala and Asn680Ser distribution was consistent with Hardy-Weinberg equilibrium(P>0.05).No significant difference was found in age,basal hormone levels for different genotype groups.Logistic regression analysis results revealed that patients with Ser680Ser genotype have a higher risk of requiring a high dose of Gn compared with patients with Asn680Asn genotype,while polymorphism of Thr307 Ala has no such effect.Conclusion:This study suggested that FSHR genotype Asn680Ser would be helpful in determining the dosage of Gn in COH;patients with Ser680Ser genotype may require higher dose of Gn.展开更多
Preimplantation genetic testing (PGT) is a widely adopted screening method that can be performed to identify and select embryos with normal ploidy;however, PGT relies on embryo biopsy, that is, polar body, embryo cell...Preimplantation genetic testing (PGT) is a widely adopted screening method that can be performed to identify and select embryos with normal ploidy;however, PGT relies on embryo biopsy, that is, polar body, embryo cells, or trophectoderm biopsy, to obtain embryonic DNA, increase its technical limitations. Studies have indicated that biopsy may have an influence on the quality and development of embryos, and increase the chance of abnormal epigenetic modifications. Therefore, non-invasive PGT (niPGT) detection of cell-free DNA (cfDNA) has gradually become a hot research topic in the field of assisted reproduction. Studies showed cfDNA could be detected in blastocyst fluid and spent culture medium (SCM) in vitro cultured embryos. The cfDNA collection requires less skill and makes lower risk to embryos. Some studies have been conducted to evaluate the feasibility of SCM-based niPGT approaches. When comparing the ploidy consistency of cfDNA in SCM, its consistency to the conventional PGT for aneuploidies results fluctuated widely, it is critical to recognize the factors influencing accuracy. These contradictory results may be related to factors such as the difference in SCM sampling methods and sampling time, and the definition of consistency. In this review, we aimed to comprehensively summarize how researchers use embryonic cfDNA to conduct niPGT detection. It also systematically reviews the factors affecting the accuracy of the test and its underlying issues, as well as prospective applications. We hope to provide a basis for future niPGT research and a useful reference for the standardized operation of niPGT that can be widely applied in clinical practice.展开更多
Male infertility is a complex reproductive disorder that impedes a huge number of couples from having children naturally in the world(Agarwal et al.,2021).As an important pathogenic factor of male infertility,spermato...Male infertility is a complex reproductive disorder that impedes a huge number of couples from having children naturally in the world(Agarwal et al.,2021).As an important pathogenic factor of male infertility,spermatogenic impairments are mainly characterized by impaired male gamete production,reduced sperm quality,or function(Tournaye et al.,2017).Spermatogenesis is a delicate and complex biological process that requires the collaboration of a large number of proteins performing different biological functions(Liu et al.,2021).展开更多
Phospholipase C zeta(PLC)is a key sperm-borne oocyte-activating factor that triggers Ca^(2+)oscillations and the subsequent block to polyspermy following gamete fusion.Mutations in PLCZ1,the gene encoding PLCζ,cause ...Phospholipase C zeta(PLC)is a key sperm-borne oocyte-activating factor that triggers Ca^(2+)oscillations and the subsequent block to polyspermy following gamete fusion.Mutations in PLCZ1,the gene encoding PLCζ,cause male infertility and intracytoplasmic sperm injection(ICSI)fertilization failure;and PLCζ expression and localization patterns are significantly correlated with ICSI fertilization rate(FR).However,in conventional in vitro fertilization(cIVF),whether and how sperm PLCζ affects fertilization remain unclear.Herein,we identified one previously reported and two novel PLCZ1 mutations associated with polyspermy in vitro that are characterized by excessive sperm-zona binding and a delay in pronuclei(PN)formation.Immunofluorescence staining and oocyte activation testing revealed that virtually all spermatozoa from patients lacked functional PLCζ and were thus unable to evoke Ca^(2+) oscillations.ICSI with an artificial oocyte activation treatment successfully rescued the polyspermic phenotype and resulted in a live birth.Furthermore,we analyzed PLCζ in an additional 58 males after cIVF treatment in the Reproductive and Genetic Hospital of CiTiC-Xiangya(Changsha,China)between February 2019 and January 2022.We found that the proportion of spermatozoa that expressed PLCζ was positively correlated with both 2PN rate and total FR.The optimal cutoff value below which males were likely to experience low FR(total FR≤30%)after clVF was 56.7%for the proportion of spermatozoa expressing PLC5.Our study expands the mutation and the phenotypic spectrum of PLCZ1 and further suggests that PLCζ constitutes a promising biomarker for identifying low FRs cases in cIVF due to sperm-related oocyte activation deficiency and that sperm PLCζ analysis may benefit the widermale population and not onlymen with IcsI failure.展开更多
Male infertility, as a major issue of human reproduction health, prevents successful natural conception. Asthenoteratospermia mainly presents one or multiple anomalies in head, neck and tail of spermatozoa, and impair...Male infertility, as a major issue of human reproduction health, prevents successful natural conception. Asthenoteratospermia mainly presents one or multiple anomalies in head, neck and tail of spermatozoa, and impairs sperm function and motility (Coutton et al., 2015). Recurrent abnormalities of the fibrous sheath lead to multiple morphological abnormaliries of the sperm flagella (MMAF), which is a quite frequent type of asthenoteratospermia in male infertility (Chemes et al., 1987;Ben Khelifa et al, 2014).展开更多
Oligoasthenoteratozoospermia(OAT)refers to the combination of various sperm abnormalities,including a decreased sperm count,reduced motility,and abnormal sperm morphology.Only a few genetic causes have been shown to b...Oligoasthenoteratozoospermia(OAT)refers to the combination of various sperm abnormalities,including a decreased sperm count,reduced motility,and abnormal sperm morphology.Only a few genetic causes have been shown to be associated with OAT.Herein,we identified a novel homozygous frameshift mutation in meiosis-specific nuclear structural 1(MNS1;NM_018365:c.603_604insG:p.Lys202Glufs*6)by whole-exome sequencing in an OAT proband from a consanguineous Chinese family.Subsequent variant screening identified four additional heterozygous MNS1 variants in 6/219 infertile individuals with oligoasthenospermia,but no MNS1 variants were observed among 223 fertile controls.Immunostaining analysis showed MNS1 to be normally located in the whole-sperm flagella,but was absent in the proband's sperm.Expression analysis by Western blot also confirmed that MNS1 was absent in the proband's sperm.Abnormal flagellum morphology and ultrastructural disturbances in outer doublet microtubules were observed in the proband's sperm.A total of three intracytoplasmic sperm injection cycles were carried out for the proband's wife,but they all failed to lead to a successful pregnancy.Overall,this is the first study to report a loss-of-function mutation in MNS1 causing OAT in a Han Chinese patient.展开更多
While it is known that spermatogonial stem cells (SSCs) initiate the production of male germ cells, the mechanisms of SSC self-renewal, proliferation, and differentiation remain poorly understood. We have previously i...While it is known that spermatogonial stem cells (SSCs) initiate the production of male germ cells, the mechanisms of SSC self-renewal, proliferation, and differentiation remain poorly understood. We have previously identified Strawberry Notch 1 (SBN01), a vertebrate strawberry notch family protein, in the proteome profile for mouse SSC maturation and differentiation, revealing SBN01 is associated with neonatal testicular development. To explore further the location and function of SBN01 in the testes, we performed Sbnol gene knockdown in mice to study the effects of SBN01 on neonatal testicular and SSC development. Our results revealed that SBN01 is required for neonatal testicular and SSC development in mice. Particularly, in vitro Sbnol gene knockdown with morpholino oligonucleotides caused a reduction of SSCs and inactivation of the noncanonical Wnt pathway, through Jun N-terminal kinases. Our study suggests SBN01 maintains SSCs by promoting the noncanonical Wnt pathway.展开更多
基金the National Natural Science Foundation for Young Scholars of China,No.82201771National Natural Science Foundation of China,No.32270912+2 种基金Natural Science Foundation of Changsha,No.kq2202491Research Grant of CITIC-Xiangya,No.YNXM202109 and No.YNXM202115Hunan Provincial Grant for Innovative Province Construction,No.2019SK4012。
文摘BACKGROUND Spermatogonial stem cells(SSCs)are the origin of male spermatogenesis,which can reconstruct germ cell lineage in mice.However,the application of SSCs for male fertility restoration is hindered due to the unclear mechanisms of proliferation and self-renewal in humans.AIM To investigate the role and mechanism of SPOC domain-containing protein 1(SPOCD1)in human SSC proliferation.METHODS We analyzed publicly available human testis single-cell RNA sequencing(RNAseq)data and found that SPOCD1 is predominantly expressed in SSCs in the early developmental stages.Small interfering RNA was applied to suppress SPOCD1 expression to detect the impacts of SPOCD1 inhibition on SSC proliferation and apoptosis.Subsequently,we explored the target genes of SPOCD1 using RNA-seq and confirmed their role by restoring the expression of the target genes.In addition,we examined SPOCD1 expression in some non-obstructive azoospermia(NOA)patients to explore the correlation between SPOCD1 and NOA.RESULTS The uniform manifold approximation and projection clustering and pseudotime analysis showed that SPOCD1 was highly expressed in the early stages of SSC,and immunohistological results showed that SPOCD1 was mainly localized in glial cell line-derived neurotrophic factor family receptor alpha-1 positive SSCs.SPOCD1 knockdown significantly inhibited cell proliferation and promoted apoptosis.RNA-seq results showed that SPOCD1 knockdown significantly downregulated genes such as adenylate kinase 4(AK4).Overexpression of AK4 in SPOCD1 knockdown cells partially reversed the phenotypic changes,indicating that AK4 is a functional target gene of SPOCD1.In addition,we found a significant downregulation of SPOCD1 expression in some NOA patients,suggesting that the downregulation of SPOCD1 may be relevant for NOA.CONCLUSION Our study broadens the understanding of human SSC fate determination and may offer new theories on the etiology of male infertility.
基金support from the National Key Research and Developmental Program of China (No.2018YFC1003603)the National Natural Science Foundation of China (No.81971445).
文摘Congenital bilateral absence of the vas deferens(CBAVD)is observed in 1%–2%of males presenting with infertility and is clearly associated with cystic fibrosis transmembrane conductance regulator(CFTR)mutations.CFTR is one of the most well-known genes related to male fertility.The frequency of CFTR mutations or impaired CFTR expression is increased in men with nonobstructive azoospermia(NOA).CFTR mutations are highly polymorphic and have established ethnic specificity.Compared with F508Del in Caucasians,the p.G970D mutation is reported to be the most frequent CFTR mutation in Chinese patients with cystic fibrosis.However,whether p.G970D participates in male infertility remains unknown.Herein,a loss-of-function CFTR p.G970D missense mutation was identified in a patient with CBAVD and NOA.Subsequent retrospective analysis of 122 Chinese patients with CBAVD showed that the mutation is a common pathogenic mutation(4.1%,5/122),excluding polymorphic sites.Furthermore,we generated model cell lines derived from mouse testes harboring the homozygous Cftr p.G965D mutation equivalent to the CFTR variant in patients.The Cftr p.G965D mutation may be lethal in spermatogonial stem cells and spermatogonia and affect the proliferation of spermatocytes and Sertoli cells.In spermatocyte GC-2(spd)ts(GC2)Cftr p.G965D cells,RNA splicing variants were detected and CFTR expression decreased,which may contribute to the phenotypes associated with impaired spermatogenesis.Thus,this study indicated that the CFTR p.G970D missense mutation might be a pathogenic mutation for CBAVD in Chinese males and associated with impaired spermatogenesis by affecting the proliferation of germ cells.
基金supported by the National Key R&D Program of China(No.2017YFC1002003)the National Natural Science Foundation of China(No.81771637 and No.81571488)+2 种基金Shanghai Collaborative Innovation Center for Translational Medicine(No.TM202015 to ZJZ)the China Postdoctoral Science Foundation(No.2019M661521)the Key Project of Research and Development of Ningxia Hui Autonomous Region of China(No.2020BFH02002).
文摘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.
基金supported by grants from the National Key Research and Development Program of China(2022YFC2702604)the National Natural Science Foundation of China(82171608,82201773,and 81971447)+1 种基金the China Postdoctoral Science Foundation(2022M711119)the Scientific Research Foundation of the Health Committee of Hunan Province(B202301039323 and B202301039518).
文摘Male infertility is a major reproductive disorder,which is clinically characterized by highly heterogeneous phenotypes of abnormal sperm count or quality.To date,five male patients with biallelic loss-of-function(LOF)variants of PARN-like ribonuclease domain-containing exonuclease 1(PNLDC1)have been reported to experience infertility with nonobstructive azoospermia.The aim of this study was to identify the genetic cause of male infertility with oligo-astheno-teratozoospermia(OAT)in a patient from a Chinese Han family.Whole-exome and Sanger sequencing analyses identified a homozygous LOF variant(NM_173516.2,c.l42C>T,p.Gln48Ter)in PNLDC1.Hematoxylin and eosin staining revealed that the spermatozoa of the patient with OAT had an irregular head phenotype,including microcephaly,head tapering,and globozoospermia.Consistently,peanut agglutinin staining of the spermatozoa revealed a complete or partial loss of the acrosome.Furthermore,the disomy rate of chromosomes in the patient’s spermatozoa was significantly increased compared with that of a fertile control sample.We reported an LOF variant of the PNLDC1 gene responsible for OAT.
基金supported by the National Natural Science Foundation of China (Nos.82070003,82100057,81900002,82101961,31970504,and 31772548)Natural Science Foundation of Hunan Province,China (Nos.2020JJ5805 and 2021JJ30943)+1 种基金Xiangya Clinical Big Data System Construction Project in Pulmonary Inflammatory Disease of Central South Universitythe National Key Clinical Specialty Construction Projects of China。
文摘Primary ciliary dyskinesia(PCD)is a congenital,motile ciliopathy with pleiotropic symptoms.Although nearly 50 causative genes have been identified,they only account for approximately 70%of definitive PCD cases.Dynein axonemal heavy chain 10(DNAH10)encodes a subunit of the inner arm dynein heavy chain in motile cilia and sperm flagella.Based on the common axoneme structure of motile cilia and sperm flagella,DNAH10 variants are likely to cause PCD.Using exome sequencing,we identified a novel DNAH10 homozygous variant(c.589C>T,p.R197W)in a patient with PCD from a consanguineous family.The patient manifested sinusitis,bronchiectasis,situs inversus,and asthenoteratozoospermia.Immunostaining analysis showed the absence of DNAH10 and DNALI1 in the respiratory cilia,and transmission electron microscopy revealed strikingly disordered axoneme 9+2 architecture and inner dynein arm defects in the respiratory cilia and sperm flagella.Subsequently,animal models of Dnah10-knockin mice harboring missense variants and Dnah10-knockout mice recapitulated the phenotypes of PCD,including chronic respiratory infection,male infertility,and hydrocephalus.To the best of our knowledge,this study is the first to report DNAH10 deficiency related to PCD in human and mouse models,which suggests that DNAH10 recessive mutation is causative of PCD.
基金This work was supported by the National Key Research and Development Program of China(2022YFC2702702 to M.L.and 2022YFC2702604 to Y.T.)the National Natural Science Foundation of China(32270899 and 32070842 to M.L.,82101961,82171608,82201773 and 81971447 to Y.T.)+1 种基金the Natural Science Foundation of Jiangsu Province(BK20190081 to M.L.)Changsha Municipal Natural Science Foundation(kq2202493 to S.Y.).We sincerely appreciate Zhuang Xiao and Shuqin Zhao from State Key Laboratory of Reproductive Medicine,Nanjing Medical University,Chen Tan,Huan Zhang,Chaofeng Tu,Qinwei Zhou,Fei Meng,Qianjun Zhang,and Chunbo Xie from Reproductive and Genetic Hospital of CITIC-XIANGYA for their assistance in this study.
文摘Dear Editor,Male subfertility,a multifactorial disease affecting~7%of the global male population,is usually caused by abnormalities in sperm flagella.The flagella and motile cilia have similar“9+2”axonemes and are evolutionarily conserved,being widely distributed in bacteria,archaea and eukaryotes1.Cilia defects also lead to primary ciliary dyskinesia(PCD),which affects approximately 1/10,000 individuals worldwide.
基金This work was supported by the grants from the National Nature Science Foundation of China (No. 81170611) and the National Nature Science Foundation of Jiangsu Province (No. BK2012601).
基金The work was supported by Graduate Independent Innovation Project Fund of Central South University(2020zzts229).
文摘Sperm morphology was once believed as one of the most predictive indicators of pregnancy outcome in assisted reproductive technology(ART).However,the impact of teratozoospermia on in vitro fertilization(IVF)/intracytoplasmic sperm injection(ICSI)outcomes and its offspring remains inconclusive.In order to evaluate the influence of teratozoospermia on pregnancy outcome and newborn status after IVF and ICSI,a retrospective study was conducted.This was a matched case-control study that included 2202 IVF cycles and 2574 ICSI cycles and was conducted at the Reproductive and Genetic Hospital of CITIC-Xiangya in Changsha,China,from June 2013 to June 2018.Patients were divided into two groups based on sperm morphology:teratozoospermia and normal sperm group.The pregnancy outcome and newborn outcome were analyzed.The results indicated that couples with teratozoospermia had a significantly lower optimal embryo rate compared to those with normal sperm morphology in IVF(P=0.007),while there were no statistically significant differences between the two groups in terms of the fertilization rate,cleavage rate,implantation rate,and pregnancy rate(all P>0.05).Additionally,teratozoospermia was associated with lower infant birth weight in multiple births after IVF.With regard to ICSI,there was no significant difference in both pregnancy outcome and newborn outcome between the teratozoospermia and normal groups(both P>0.05).Furthermore,no increase in the risk of birth defects occurred in the teratozoospermia group after IVF/ICSI.Consequently,we believe that teratozoospermia has limited predictive value for pregnancy outcomes in IVF/ICSI,and has little impact on the resulting offspring if multiple pregnancy is avoided.
基金support of the National Key Research&Developmental Program of China(2018YFC1004900 to YQT)the National Natural Science Foundation of China(81971447 to YQT)the Key Grant of Prevention and Treatment of Birth Defect from Hunan province(2019SK1012 to YQT),and the research grant ofCITIC-Xiangya(YNXM-202004,YNXM-202006).
文摘Spermiogenesis is a complex and tightly regulated process,consisting of acrosomal biogenesis,condensation of chromatin,flagellar assembly,and disposal of extra cytoplasm.Previous studies have reported that sperm flagellar 2(SPEF2)deficiency causes severe asthenoteratozoospermia owing to spermiogenesis failure,but the underlying molecular mechanism in humans remains unclear.Here,we performed proteomic analysis on spermatozoa from three SPEF2 mutant patients to study the functional role of SPEF2 during sperm tail development.A total of 1262 differentially expressed proteins were detected,including 486 upregulated and 776 downregulated.The constructed heat map of the differentially expressed proteins showed similar trends.Among these,the expression of proteins related to flagellar assembly,including SPEF2,sperm associated antigen 6(SPAG6),dynein light chain tctex-type 1(DYNLT1),radial spoke head component 1(RSPH1),translocase of outer mitochondrial membrane 20(TOM20),EF-hand domain containing 1(EFHC1),meiosis-specific nuclear structural 1(MNS1)and intraflagellar transport 20(IFT20),was verified by western blot.Functional clustering analysis indicated that these differentially expressed proteins were specifically enriched for terms such as spermatid development and flagellar assembly.Furthermore,we showed that SPEF2 interacts with radial spoke head component 9(RSPH9)and IFT20 in vitro,which are well-studied components of radial spokes or intra-flagellar transport and are essential for flagellar assembly.These results provide a rich resource for further investigation into the molecular mechanism underlying the role that SPEF2 plays in sperm tail development and could provide a theoretical basis for gene therapy in SPEF2 mutant patients in the future.
基金This study was supported by grants from the National Key Research&Developmental Program of China(2018YFC1004901)National Natural Science Foundation of China(81771645 and 81971447)+2 种基金Hunan Provincial Natural Science Foundation of China(2019JJ51006)the Key Grant of Prevention and Treatment of Birth Defect from Hunan Province(2019SK1012)the Scientific Research Foundation of Reproductive and Genetic Hospital of CITIC-Xiangya grants(YNXM-201912).
文摘Diminished ovarian reserve(DOR)is a disorder of ovarian function in which the ovary loses its normal reproductive potential,including decreasing oocyte quantity and quality.The disorder is associated with infertility in women of reproductive age with regular menses and poor response to controlled ovarian hyperstimulation(Broekmans et al.,2009).
基金This study was supported by the Hunan Province Municipal Natural Science Foundation(2022JJ30018)to WNLHunan Province Health Commission Science Foundation(B202301037899)to WNLResearch Grant of CITIC-Xiangya(YNXM-201904)to WNL.
文摘The clinical applications of acrosin activity are limited.We analyzed 61578 male partners in infertile couples who visited the outpatient department of the Reproductive and Genetic Hospital of CITIC-Xiangya(Changsha,China)between August 2014 and December 2019 to determine the reference ranges and thresholds for acrosin activity in infertile Chinese men;to determine whether correlations exist between acrosin activity and age,sperm concentration,sperm morphology,or sperm motility;and to evaluate whether acrosin activity could serve as an effective prognostic indicator for choosing between in vitro fertilization(IvF)and intracytoplasmic sperm injection(icsl)in the clinic.The cut-off value for the normal reference range of acrosin activity for male partners in infertile couples was 24.78μlU per 106 sperm.There was no significant association between acrosin activity and age,sperm concentration,semen volume,total sperm count,progressive motility,or total motile spermatozoa.A weak positive correlation was found between acrosin activity and normal sperm morphology.There was a statistically significant difference in abnormal acrosome morphology between the group with high acrosin activity(>24.78μlU per 106 sperm)and the group with low acrosin activity(<24.78μlU per 106 sperm).The group with a low IVF fertilization rate had a high index of abnormal acrosomal morphology at 21.2%,while the group with a high IVF fertilization rate had a low index of 0.2%.At an acrosin activity of<24.78μlU per 10 sperm,in one cycle of the same patient,the fertilization rate,normal fertilization rate,and good-quality embryo rate for Icsl were significantly higher than those for IVF.Therefore,the most promising application of acrosin activity could be in the selection of IcsI over IVF for infertile male patients with complete fertilization failure or a low fertilization rate.
基金financially supported by Province Funds of Zhejiang University Medical School Key Laboratory(2012-RG/GH-0006)Jiangsu Key talents of maternal and child health(FRC2017250)+1 种基金Clinical research special funds of Wu Jieping Foundation,China(320.6755.15027)Suzhou Key Medical Center,(SZZX201505).
文摘Objective:This study is to investigate the effect of different single-nucleotide polymorphisms of follicle-stimulating hormone receptor(FSHR)gene on gonadotropin(Gn)administration dosage during controlled ovarian hyperstimulation(COH)protocol of in vitro fertilization and embryo transfer.Methods:This retrospective study included 184 Chinese infertile women in Center for Reproduction and Genetics of Suzhou Municipal Hospital from June 2012 to 2014.All of the enrolled patients were homogeneous in some basal characteristics,and they all met the eligibility criteria.Blood tests were conducted on day 3 of menstrual cycle or the day of human chorionic gonadotropin administration for hormonal profile analysis and DNA extraction.DNA sequencing was performed for polymorphism analysis.The participants were classified into threonine(Thr)/Thr,Thr/alanine(Ala),and Ala/Ala groups according to genotype at position 307,and asparagine/asparagine(Asn/Asn),Asn/serine(Ser),and Ser/Ser groups according to genotype at position 680.Logistic regression and correlation analyses were performed to identify the effect of these two polymorphisms on Gn consumption.Results:The frequency of Thr307Ala and Asn680Ser distribution was consistent with Hardy-Weinberg equilibrium(P>0.05).No significant difference was found in age,basal hormone levels for different genotype groups.Logistic regression analysis results revealed that patients with Ser680Ser genotype have a higher risk of requiring a high dose of Gn compared with patients with Asn680Asn genotype,while polymorphism of Thr307 Ala has no such effect.Conclusion:This study suggested that FSHR genotype Asn680Ser would be helpful in determining the dosage of Gn in COH;patients with Ser680Ser genotype may require higher dose of Gn.
基金This work was supported by the Science and Technology Major Projects of Birth Defect Collaborative Prevention in Hunan Province(No.1019SK1010).
文摘Preimplantation genetic testing (PGT) is a widely adopted screening method that can be performed to identify and select embryos with normal ploidy;however, PGT relies on embryo biopsy, that is, polar body, embryo cells, or trophectoderm biopsy, to obtain embryonic DNA, increase its technical limitations. Studies have indicated that biopsy may have an influence on the quality and development of embryos, and increase the chance of abnormal epigenetic modifications. Therefore, non-invasive PGT (niPGT) detection of cell-free DNA (cfDNA) has gradually become a hot research topic in the field of assisted reproduction. Studies showed cfDNA could be detected in blastocyst fluid and spent culture medium (SCM) in vitro cultured embryos. The cfDNA collection requires less skill and makes lower risk to embryos. Some studies have been conducted to evaluate the feasibility of SCM-based niPGT approaches. When comparing the ploidy consistency of cfDNA in SCM, its consistency to the conventional PGT for aneuploidies results fluctuated widely, it is critical to recognize the factors influencing accuracy. These contradictory results may be related to factors such as the difference in SCM sampling methods and sampling time, and the definition of consistency. In this review, we aimed to comprehensively summarize how researchers use embryonic cfDNA to conduct niPGT detection. It also systematically reviews the factors affecting the accuracy of the test and its underlying issues, as well as prospective applications. We hope to provide a basis for future niPGT research and a useful reference for the standardized operation of niPGT that can be widely applied in clinical practice.
基金supported by the National Key Research and Development Program of China(2021YFC2701400 and 2021YFC2700901)the National Natural Science Foundation of China(32100480,82171607,and 81971441)+4 种基金the Shanghai Municipal Science and Technology Major Project(2017SHZDZX01)the Scientific Research(TP202002)from Anhui Medical Universitythe China Postdoctoral Science Foundation(2020TQ0072)the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences(2019PT310002)supported by Shanghai Municipal Commission for Science and Technology Grants(19411951800)。
文摘Male infertility is a complex reproductive disorder that impedes a huge number of couples from having children naturally in the world(Agarwal et al.,2021).As an important pathogenic factor of male infertility,spermatogenic impairments are mainly characterized by impaired male gamete production,reduced sperm quality,or function(Tournaye et al.,2017).Spermatogenesis is a delicate and complex biological process that requires the collaboration of a large number of proteins performing different biological functions(Liu et al.,2021).
基金supported by grants from the National Natural Science Foundation of China(No.81901553 to CD)the Natural Science Foundation of Hunan Province,China(No.2021JJ30461 to CD).
文摘Phospholipase C zeta(PLC)is a key sperm-borne oocyte-activating factor that triggers Ca^(2+)oscillations and the subsequent block to polyspermy following gamete fusion.Mutations in PLCZ1,the gene encoding PLCζ,cause male infertility and intracytoplasmic sperm injection(ICSI)fertilization failure;and PLCζ expression and localization patterns are significantly correlated with ICSI fertilization rate(FR).However,in conventional in vitro fertilization(cIVF),whether and how sperm PLCζ affects fertilization remain unclear.Herein,we identified one previously reported and two novel PLCZ1 mutations associated with polyspermy in vitro that are characterized by excessive sperm-zona binding and a delay in pronuclei(PN)formation.Immunofluorescence staining and oocyte activation testing revealed that virtually all spermatozoa from patients lacked functional PLCζ and were thus unable to evoke Ca^(2+) oscillations.ICSI with an artificial oocyte activation treatment successfully rescued the polyspermic phenotype and resulted in a live birth.Furthermore,we analyzed PLCζ in an additional 58 males after cIVF treatment in the Reproductive and Genetic Hospital of CiTiC-Xiangya(Changsha,China)between February 2019 and January 2022.We found that the proportion of spermatozoa that expressed PLCζ was positively correlated with both 2PN rate and total FR.The optimal cutoff value below which males were likely to experience low FR(total FR≤30%)after clVF was 56.7%for the proportion of spermatozoa expressing PLC5.Our study expands the mutation and the phenotypic spectrum of PLCZ1 and further suggests that PLCζ constitutes a promising biomarker for identifying low FRs cases in cIVF due to sperm-related oocyte activation deficiency and that sperm PLCζ analysis may benefit the widermale population and not onlymen with IcsI failure.
基金supported by National Natural Science Foundation of China (31625015 and 31521003)Foundation of the Education Department of Anhui Province (KJ2016A370)+1 种基金Foundation of the Department of Science and Technology of Anhui Province (2017070802D150)Shanghai Municipal Science and Technology Major Project (2017SHZDZX01)
文摘Male infertility, as a major issue of human reproduction health, prevents successful natural conception. Asthenoteratospermia mainly presents one or multiple anomalies in head, neck and tail of spermatozoa, and impairs sperm function and motility (Coutton et al., 2015). Recurrent abnormalities of the fibrous sheath lead to multiple morphological abnormaliries of the sperm flagella (MMAF), which is a quite frequent type of asthenoteratospermia in male infertility (Chemes et al., 1987;Ben Khelifa et al, 2014).
基金This study was supported by grants from the National Key Science Program S&T Program(2018YFC1004900 to YQT)the National Natural Science Foundation of China(81971447 to YQT)+1 种基金the Science and Technology Major Project of the Ministry of Science and Technology of Hunan Province(2017SK1030 to YQT)Graduate Research and Innovation Projects of Central South University(2019zzts734 to YL and 2019zzts322 to WLW)。
文摘Oligoasthenoteratozoospermia(OAT)refers to the combination of various sperm abnormalities,including a decreased sperm count,reduced motility,and abnormal sperm morphology.Only a few genetic causes have been shown to be associated with OAT.Herein,we identified a novel homozygous frameshift mutation in meiosis-specific nuclear structural 1(MNS1;NM_018365:c.603_604insG:p.Lys202Glufs*6)by whole-exome sequencing in an OAT proband from a consanguineous Chinese family.Subsequent variant screening identified four additional heterozygous MNS1 variants in 6/219 infertile individuals with oligoasthenospermia,but no MNS1 variants were observed among 223 fertile controls.Immunostaining analysis showed MNS1 to be normally located in the whole-sperm flagella,but was absent in the proband's sperm.Expression analysis by Western blot also confirmed that MNS1 was absent in the proband's sperm.Abnormal flagellum morphology and ultrastructural disturbances in outer doublet microtubules were observed in the proband's sperm.A total of three intracytoplasmic sperm injection cycles were carried out for the proband's wife,but they all failed to lead to a successful pregnancy.Overall,this is the first study to report a loss-of-function mutation in MNS1 causing OAT in a Han Chinese patient.
基金the Suzhou Key Medical Center (grant number SZZX201505)the Jiangsu Provincial Medical Innovation Team (grant number CXTDB2017013)+5 种基金the Suzhou Introduced Project of Clinical Medical Expert Team (grant number SZYJTD201708)the National Natural Science Foundation of China (grant number 31701298)the Natural Science Foundation of Jiangsu Province (grant number 20170562)the Key Research Fund for Zhenjiang Social Development (grant number SH2016028)the Key Research Fund for Zhenjiang Health Science and Technology (grant number SHW2016001)the Open Fund of State Key Laboratory of Reproductive Medicine of Nanjing Medical University (grant numbers SKLRM-KA201603, SKLRM-KA201704).
文摘While it is known that spermatogonial stem cells (SSCs) initiate the production of male germ cells, the mechanisms of SSC self-renewal, proliferation, and differentiation remain poorly understood. We have previously identified Strawberry Notch 1 (SBN01), a vertebrate strawberry notch family protein, in the proteome profile for mouse SSC maturation and differentiation, revealing SBN01 is associated with neonatal testicular development. To explore further the location and function of SBN01 in the testes, we performed Sbnol gene knockdown in mice to study the effects of SBN01 on neonatal testicular and SSC development. Our results revealed that SBN01 is required for neonatal testicular and SSC development in mice. Particularly, in vitro Sbnol gene knockdown with morpholino oligonucleotides caused a reduction of SSCs and inactivation of the noncanonical Wnt pathway, through Jun N-terminal kinases. Our study suggests SBN01 maintains SSCs by promoting the noncanonical Wnt pathway.