STUDY QUESTION:What are the prevalence, type, and stratified risks (reproductive failure subtype, sex, age, semen quality, and prior adverse pregnancy events) of chromosomal aberrations in couples seeking ART treatment in a large-scale cohort study? SUMMARY ANSWER:The prevalence of chromosomal aberrations in couples with reproductive failure was 3.42%, with a higher prevalence in younger individuals, men with poorer semen quality, and those experiencing multiple adverse pregnancy outcomes. WHAT IS KNOWN ALREADY:Reproductive failure is a global health issue, with chromosomal aberrations playing an important role. ART is widely used for the treatment of reproductive failure; however, large-scale, comprehensive studies characterizing the stratified risks of chromosomal aberrations in couples seeking ART remain scarce. STUDY DESIGN, SIZE, DURATION:This retrospective study analysed chromosomal data from 227 818 couples seeking ART at our hospital between January 1993 and March 2024. PARTICIPANTS/MATERIALS, SETTING, METHODS:This study included 227 818 couples with reproductive failure, including 130 213 couples with primary infertility, 58 161 with secondary infertility, and 39 444 with adverse pregnancy outcomes. All participants underwent routine cytogenetic analysis using GTG-banding prior to ART. Statistical analysis was performed using R software (v4.4.0), with significance set at P < 0.05. MAIN RESULTS AND THE ROLE OF CHANCE:Chromosomal aberrations were detected in 7785 couples (3.42%), with the highest prevalence in those with adverse pregnancy outcomes (4.83%), followed by those with primary infertility (3.54%) or secondary infertility (2.18%) (all P < 0.001). Significant sex differences were observed in infertile couples (men: 1.91% vs women: 1.52%) and couples with adverse pregnancy outcomes (men: 2.04% vs women: 2.81%), (P < 0.001). The prevalence of chromosomal aberrations was inversely correlated with age (2.36% in <25 years to 1.29% in ≥35 years) and increased significantly with poorer semen quality (0.89% in normozoospermia to 12.54% in azoospermia) and more adverse pregnancy events (3.07% in 1 event to 9.38% in ≥3 events, P < 0.001). Common structural aberrations included reciprocal and Robertsonian translocations and inversions, with frequent breakpoints at 11q23, 22q11, and 7q22. The percentage of haploid length of each autosome and the corresponding percentage of breakpoints showed a strong Pearson's correlation (R = 0.933, P < 0.001, two-tailed test). Robertsonian translocations and t(11;22)(q23;q11) were recurrent. The most frequent aneuploidies identified were 47,XXY (Klinefelter) and 45, X (Turner) syndromes, in both mosaic and non-mosaic forms. LIMITATIONS, REASONS FOR CAUTION:The lack of detailed clinical characteristics limited patient stratification and hindered the in-depth analysis of karyotype-phenotype relationships. The absence of a large fertile control group restricted comparative analysis. Conventional GTG-banding resolution may miss some cryptic chromosomal abnormalities. Single-centre data may limit the generalizability of our findings to more diverse populations. WIDER IMPLICATIONS OF THE FINDINGS:Stratified risk data (such as the higher prevalence of chromosomal aberrations in younger populations, individuals with poor semen quality, or those with a history of multiple adverse pregnancies) provide crucial evidence for clinicians to assess reproductive risks associated with chromosomal aberrations and make karyotyping decisions prior to ART treatment. STUDY FUNDING/COMPETING INTEREST(S):This work was supported by the National Key Research and Development Program of China (2023YFC2705605). The authors declare that there are no competing interests. TRIAL REGISTRATION NUMBER:N/A.

STUDY QUESTION:What is the genetic etiology of recurrent blastulation failure, particularly in morphologically good-quality cleavage-stage embryos? SUMMARY ANSWER:Variants in meiotic genes may contribute to gamete-derived complex aneuploidy and impaired embryonic genome activation, which are strongly associated with recurrent blastulation failure in good-quality cleavage-stage embryos (R-GQBF). WHAT IS KNOWN ALREADY:Successful blastocyst formation is critical for implantation. Embryonic development undergoes a major transition around the 8‑cell stage, shifting from reliance on maternal transcripts to embryonic genome activation. While pathogenic variants in maternal-effect genes have been linked to developmental arrest before the 8-cell stage, the genetic basis of failure occurring between the 8-cell stage and blastulation remains unclear. STUDY DESIGN SIZE DURATION:From 2018 to 2023, 707 couples who met the R-GQBF criteria were recruited. After rigorous exclusion, 204 couples remained, of whom 97 were ultimately included for genetic etiology analysis. PARTICIPANTS/MATERIALS SETTING METHODS:A total of 109 individuals from 97 selected couples (93 females and 16 males, including 13 couples with both partners) underwent whole-exome sequencing (WES). Genetic variants were compared with those from 1000 fertile controls (500 females and 500 males). The chromosomal constitutions of 103 blastulation-failure embryos from 50 R-GQBF couples were analyzed via WES. Copy number variation (CNV) parental origin analysis was performed on 13 aneuploid embryos. Paternally derived chromosomal anomalies were further investigated using single-sperm chromosome analysis. Additionally, single-cell RNA sequencing was carried out on 15 arrested embryos. MAIN RESULTS AND THE ROLE OF CHANCE:Twenty-five variants in 10 meiotic genes were identified in 20 patients (18.3%). Female carriers predominantly harbored prophase I variants (SPO11, MEI1, REC114, ANKRD31, DMC1, CNTD1, MLH3, SYCE1, and SYCP2), while three male patients carried MEIKIN variants. Female carriers generally had preserved ovarian reserve, whereas male carriers showed severe oligoasthenoteratozoospermia. Chromosomal analysis revealed a high prevalence of complex aneuploidy in blastulation-failure embryos (63.1%). CNV tracing confirmed the parental origin of abnormalities from the meiotic variant carrier in analyzed case, and sperm from all MEIKIN carriers also exhibited severe chromosomal abnormalities. Single-cell transcriptomics revealed defective embryonic genome activation, impaired lineage specification, and activation of stress pathways. These consistent findings minimize the likelihood of chance associations. LARGE SCALE DATA:N/A. LIMITATIONS REASONS FOR CAUTION:Direct evaluation of oocyte chromosomal integrity was not feasible. Larger cohorts may reveal additional genes, and functional studies in animal models are required to validate genotype-phenotype relationships. WIDER IMPLICATIONS OF THE FINDINGS:This study identifies biparental meiotic variants as an underrecognized cause of R-GQBF. Clinically, beyond routine extended embryo culture, chromosomal analysis should be recommended for etiological investigation. For couples experiencing recurrent complex aneuploidy in good-quality cleavage-stage embryos, genetic screening for meiotic variants may aid in diagnosis, counseling, and personalized treatment planning. STUDY FUNDING/COMPETING INTERESTS:This work was supported by the National Natural Science Foundation of China (32270911, 82371672, 82471697, 62588301), the Natural Science Foundation of Hunan Province (2024JJ2083, 2023JJ30714, 2023JJ10084), the Science and Technology Innovation Program of Hunan Province (2023RC3233), the Fundamental and Interdisciplinary Disciplines Breakthrough Plan of the Ministry of Education of China (JYB2025XDXM117), the National Key R&D Program of China (2023YFC2705504), and the project of the Reproductive and Genetic Hospital of CITIC-XIANGYA (YNXM-202221, YNXM-202402, and YNXM-202505). The authors declare no competing interests.

High levels of sperm DNA fragmentation index (DFI) represent a critical factor in male infertility, with detrimental effects on embryonic development and offspring well-being. However, selecting sperm with low DFI remains a tremendous challenge. Here, we present an organ-level selection strategy capable of isolating spermatozoa with ultra-low DFI, achieving a remarkable reduction to 0.13% compared to 34.57% observed in raw semen. We design and fabricate a female reproductive tract (FRT)-on-a-chip (FRToC) device that mimics the entire physiological microenvironment for in vivo sperm selection, with clinical validation performed using samples from patients. The FRToC selects sperm with ultra-low DFI (mean: 0.71%) from patients with high DFI (mean: 41.93%), while also ensuring superior sperm motility and acrosome integrity. Additionally, trace sperm proteomic and single-cell copy number variants (CNV) analyses revealed that sperm sorted by FRToC exhibited an increased capacity to mitigate oxidative stress, thus resulting in more intact chromosomes. Our organ-scale selection method underscores the potential of the FRToC to select high-quality spermatozoa, offering a promising improvement for assisted reproductive technology (ART).

Human mesenchymal stromal cells (MSCs) possess extensive therapeutic potential, and their biological characteristics and functions can be modulated by culture media. This study aimed to systematically evaluate the effects of two basal media, DMEM/F12(DF12) and low-glucose Dulbecco's Modified Eagle Medium (LG-DMEM), on the biological characteristics and functions of umbilical cord-derived MSCs. The influences of basal medium on crucial quality parameters of MSCs, including identity, differentiation potential, proliferation, replicative senescence, genetic stability, gene expression profile, and immunomodulatory capacity, were assessed. MSCs cultured in DF12 exhibited smaller cell size, faster proliferation, higher expression levels of genes associated with growth factor activity, cytokine activity, and extracellular matrix binding, but showed more notable replicative senescence with serial passages. MSCs cultured in LG-DMEM expressed higher levels of indoleamine 2,3-dioxygenase (IDO), had a stronger inhibitory effect on T cell proliferation in vitro, especially on Th17 cells, and showed a better therapeutic effect on alleviating skin lesions in imiquimod (IMQ)-induced psoriasis-like mice. These results suggest that DF12 is more suitable for the early passages of MSCs, potentially favoring applications in injury repair, while LG-DMEM is more suitable for the culture of higher passages of MSCs and enhances efficacy in immune-related diseases. These findings provide insights for selecting the appropriate basal media, a critical process parameter during expansion, contributing to the optimization of MSCs manufacturing bioprocesses to meet the requisite quantity and quality for therapeutic applications.

Congenital cataract is a vision impairment that arises before birth or within the first year of life due to lens opacity, with genetic disorders being a significant contributing factor. The purpose of this study is to identify the pathogenic variants associated with congenital cataract to inform clinical diagnosis, treatment, reproductive intervention. Next-generation sequencing was performed on 107 families with congenital cataracts, followed by Sanger sequencing validation and familial analysis of candidate variants. RNA splicing pattern analysis was conducted on variants located near splicing sites. A total of 44 likely pathogenic or pathogenic variants (including 20 novel variants) in 46 families, and 19 variants of uncertain significance (VUS) in 19 families were identified, with a detection rate of 43.0%. A total of 14 VUS were sub-classified as "warm" or "hot" VUS based on Bayesian analysis. PGT or prenatal diagnosis was conducted in 26 families; follow-up revealed that 16 families opted for PGT and successfully delivered 10 healthy children. Among 10 families that pursued prenatal diagnosis after natural conception, 8 gave birth to healthy children.This study expanded the variant spectrum and providing valuable insights for the prevention and management of this condition.

Abstract STUDY QUESTION In best-prognosis preimplantation genetic testing (PGT) cycles—defined by the availability of multiple transferable embryos—can polygenic embryo screening (PES) provide meaningful, context-dependent stratification that may influence embryo prioritization? SUMMARY ANSWER In a sister-pair breast cancer validation cohort, the polygenic risk score (PRS) showed modest predictive performance, and when applied retrospectively to embryos, differences in modelled susceptibility were context-dependent, primarily influenced by the number of available embryos, parental polygenic risk profile, the limited discriminative power of the PRS, and the presence of disease-related monogenic variants. WHAT IS KNOWN ALREADY The use of PES as an embryo-ranking tool within PGT remains controversial. Its potential utility depends on embryo availability and clinical context, yet empirical evaluations in real-world PGT settings remain limited. STUDY DESIGN, SIZE, DURATION This retrospective study combined (i) family-based validation using 184 affected–unaffected sister pairs, and (ii) embryo-level analysis of 310 preimplantation genetic testing for monogenic disorders (PGT-M) cycles comprising 1,722 embryos. Simulation modelling estimated differences in modelled embryo-level PRS percentile ranking under varying clinical and genetic scenarios. PARTICIPANTS/MATERIALS, SETTING, METHODS Family-based performance of a breast cancer PRS was evaluated using affected and unaffected sister pairs. Embryo genotype data from PGT-M cycles were used to model PES under varying conditions, including the number of transferable embryos, the presence of pathogenic monogenic variants, and parental PRS percentile strata. MAIN RESULTS AND THE ROLE OF CHANCE Within-family PRS discrimination was modest: sisters in the top 10% of the PRS distribution had 4.07-fold higher breast cancer odds than their siblings (95% CI: 1.55–6.33). In the PGT-M cohort, 55% of cycles produced ≥3 transferable embryos, with a median within-cycle spread of approximately 25 PRS percentiles. In BRCA1/2-related cycles, PRS did not introduce distinct risk strata but revealed dispersion in modelled susceptibility among embryos sharing the same monogenic background. Embryo PRS distributions were concordant with parental profiles: 71% of embryos from couples with parental PRS ≥80th percentile were classified as high-PRS, compared with 7% when at least one parent was ≤20th percentile. Simulation analyses indicated that PRS-based embryo prioritization was associated with a 12-point lower mean embryo PRS percentile compared with morphology-based embryo prioritization in PGT-M cycles, attenuating to 10 points when PGT-A was incorporated. These differences reflect a statistical surrogate outcome only and do not demonstrate reduction in lifetime disease incidence or other clinical benefits. LARGE SCALE DATA Clinical data from 310 PGT-M cycles involving 1,722 embryos and a family-based validation cohort of 184 affected–unaffected sister pairs were analyzed. No population-scale dataset was generated. LIMITATIONS, REASONS FOR CAUTION These findings derive from high-prognosis PGT-M cycles and may not generalize to other PGT settings. Current PRS models have modest performance and variable transferability across ancestries. The retrospective design, limited subgroup sizes, and modelling assumptions (e.g., aneuploidy and sex distribution) further constrain interpretation. The primary simulation outcome—PRS percentile shift—is a hypothesis-generating surrogate endpoint, not a clinical outcome; clinical benefit was not assessed or demonstrated. These findings should not be interpreted as supporting routine clinical implementation of PES; premature use may increase parental anxiety, inequitable access, and pressure to rank embryos using predictions that remain unvalidated at the embryo level. WIDER IMPLICATIONS OF THE FINDINGS This study provides an empirical framework for evaluating the current limits of PES within PGT. The observed PRS percentile shifts appear concentrated in best-prognosis cycles with multiple transferable embryos and elevated parental PRS, whereas little change is observed when embryo numbers are limited or risk is dominated by high-penetrance monogenic variants. These findings are hypothesis-generating and may inform future prospective research and ethical discussion, while underscoring that clinical utility and implementation require further validation within appropriately governed settings. FUNDING This study was funded by Major Scientific Program of CITIC Group (No. 2023ZXKYB34100), the Science Foundation of Hunan Province (Grant 2023JJ30422), and Health Research Project of Hunan Provincial Health Commission (grant number: W20243089). DISCLOSURES The authors declare no conflicts of interest.

BackgroundSevere combined immunodeficiency (SCID) is one of the most severe forms of primary immunodeficiency. JAK3 gene is a critical determinant of SCID, as JAK3-STAT pathway regulates development, proliferation, activation, and differentiation of immune cells. This study aimed to identify the genetic cause of a family with a suspected SCID patient, and to perform carrier screening for two couples to assess the risk of conceiving offspring with birth defects.MethodsWhole-Exome Sequencing was performed on five individuals from the three families. A series of in vitro functional experiments, including Western blotting and luciferase assays, were conducted to assess the pathogenicity of the identified JAK3 variants.ResultsWe identified seven JAK3 variants, including five variants of uncertain significance (p.Arg402His, p.ILe688Phe, p.Leu129Phe, p.Met235Thr, p.Ala634Pro) and one pathogenic variant and one likely pathogenic variant (p.Gln1007Ter and p.Cys376Leufs*34). Among these, four variants (p.Gln1007Ter, p.Leu129Phe, p.Cys376Leufs*34 and p.Ala634Pro) were novel. In vitro functional experiments revealed that three of five variants of uncertain significance (VUSs) significantly reduced STAT5 phosphorylation and transcriptional activity, thereby reclassifying two variants (p.Arg402His and p.ILe688Phe) as likely pathogenic variants (LP) and one variant (p.Leu129Phe) as VUS with a Bayesian score of 3. In contrast, the remaining two variants (p.Ala634Pro and p.Met235Thr) did not affect JAK3 function, and were reclassified as VUS with a Bayesian score of 1 or 0.ConclusionThis study identified seven JAK3 variants from three families, including four novel variants. Functional experiments revealed that two VUSs were reclassified as LP and one VUS were reclassified as VUS with a Bayesian score of 3. These findings highlight the importance of integrating genetic and functional analyses to enhance diagnostic accuracy, inform treatment strategies for patients, clarify of the risk for carrier-screening couples, improve genetic counseling, and guide reproductive interventions.

To elucidate the genetic etiology of osteogenesis imperfecta (OI) in affected families and to characterize reproductive decision-making, reproductive interventions, and pregnancy outcomes of these families. Fifty-one families with clinically suspected OI underwent variant analysis by Sanger sequencing or next-generation sequencing (NGS) and clinical follow-up of reproductive options and pregnancy outcomes. Of the 51 enrolled families, 37 probands had mild phenotypes and 14 presented with severe phenotypes. Genetic analysis revealed that 46 probands (90.2

OBJECTIVE:To explore the genetic etiology of 46 Chinese pedigrees affected with Hereditary multiple exostoses (HME) and provide genetic counseling and reproductive intervention. METHODS:Whole-exome sequencing and Sanger sequencing were carried out on 87 patients from the 46 pedigrees to analyze the variants of EXT1 and EXT2 genes. Pathogenicity of the variants was assessed based on the guidelines from the American College of Medical Genetics and Genomics and Association for Molecular Pathology (ACMG/AMP). Prenatal diagnosis and preimplantation genetic testing (PGT) were provided for couples with identified pathogenic mutations. This study was approved by the Medical Ethics Committee of the hospital (Ethics No.: LL-SC-SG-2014-010). RESULTS:In total 17 and 22 pathogenic variants were respectively identified in the EXT1 and EXT2 genes, among which 5 EXT1 and 12 EXT2 variants were unreported previously. Three patients with no family history were found to harbor de novo variants of the EXT1 gene. Twenty nine couples had opted for PGT or underwent prenatal diagnosis following natural conception, and 17 healthy babies were born. CONCLUSION:This study has clarified the genetic etiology of 45 HME pedigrees and identified 17 novel variants, which has enriched the mutational spectrum of the EXT1 and EXT2 genes. Reproductive intervention through PGT and prenatal diagnosis have prevented the recurrence of HME in these families.

OBJECTIVE:To explore the genetic etiology of 210 Chinese pedigrees affected with Hemophilia and provide prenatal diagnosis and preimplantation genetic testing (PGT) for them. METHODS:A total of 210 unrelated pedigrees diagnosed with Hemophilia (176 with Hemophilia A and 34 with Hemophilia B) at the Reproductive and Genetic Hospital of CITIC-Xiangya between 2011 and 2024 were selected as study subjects. Genetic testing was carried out on the probands, including whole-exome sequencing, PCR-Sanger sequencing, and detection of intron 22 and intron 1 inversions of the F8 gene. Pathogenicity of candidate variants was classified based on the guidelines from the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP). After the determination of genetic causes, reproductive intervention was implemented through prenatal diagnosis or preimplantation genetic testing (PGT). This study was approved by the Medical Ethics Committee of the hospital (Ethics No.: LL-SC-SG-2014-010). RESULTS:In total 137 pathogenic or likely pathogenic variants of the F8 and F9 genes were identified among 207 pedigrees (detection rate = 98.5%), among which 16 were were unreported previously. Respectively, 105 pathogenic or likely pathogenic variants (including 13 novel ones) of the F8 gene were detected among 174 pedigrees with Hemophilia A, while 32 pathogenic or likely pathogenic variants (including 3 novel ones) of the F9 gene were identified among 33 pedigrees with Hemophilia B. Among the 207 pedigrees, 65 had opted for prenatal diagnosis, and 9 fetuses were diagnosed with Hemophilia, and the pregnancies were terminated. On the other hand, 71 pedigrees had opted PGT, with 454 embryos from 90 cycles completed testing, which yielded a diagnostic rate of 99.8% (453/454). As a result, 100 healthy infants were born through reproductive intervention. CONCLUSION:This study has clarified the genetic basis of 207 pedigrees affected with Hemophilia. The results had expanded the mutational spectrum, and reduced the birth rate of Hemophilia by prenatal diagnosis and PGT.

Accurate identification of the genetic determinants of rare diseases is essential for effective recurrence-risk management and informed reproductive decision-making. Although whole-exome sequencing (WES) and whole-genome sequencing (WGS) have significantly improved diagnostic capabilities, a subset of affected families still receives no definitive molecular diagnosis. RNA sequencing (RNA-seq) has emerged as a promising complementary diagnostic tool, yet its clinical implementation in the context of preconception genetic counseling remains underexplored. We used phytohemagglutinin-activated peripheral blood cells (PHACs) as a robust RNA source and enhanced conventional RNA-seq through the integration of three analytical innovations: (1) transcript isoform distribution (TID) analysis, (2) realignment against the MANE (Matched Annotation from NCBI and EMBL-EBI) reference transcriptome, and (3) pharmacological induction-based cryptic splicing detection. This optimized pipeline was applied to 55 rare-disease families with negative WES/WGS results who were undergoing preconception genetic counseling. Based on prior evaluations, families were grouped as VUS (n = 7), suspected-gene/variant-negative (n = 10), and unsolved/no-candidate (n = 38). PHACs showed reduced interindividual variability and higher RNA integrity than fresh PBMCs (median RIN: 9.77 vs. 8.97; p < 0.0001). The optimized workflow improved diagnostic yield by 2.2-fold (20% vs. 9%). Stratified analysis revealed positive rates of 71% (VUS), 40% (suspected-gene/variant-negative), and 5.2% (unsolved/no-candidate). Among the 11 positive cases, 10 received definitive diagnoses, leading to diverse reproductive decisions. This enhanced RNA-seq workflow provides a clinically applicable and scalable strategy for improving molecular diagnostics in reproductive and preconception settings, offering a valuable model for future clinical transcriptomics.

BackgroundAcute liver failure (ALF) is a life-threatening syndrome characterized by rapid deterioration of liver function, resulting in high mortality and posing a substantial global health burden. Human embryonic stem cells (hESCs) possess unlimited self-renewal capacity and pluripotent differentiation potential. Transplantation of hESC-derived hepatocyte-like cells (HPLCs) represents a promising therapeutic strategy for ALF.MethodsA good manufacturing practice (GMP)-compliant differentiation process was developed to generate HPLCs from hESCs, and their biological characteristics and functional properties were systematically evaluated. A comprehensive series of preclinical safety and efficacy studies was performed, including dose-escalation experiments, biodistribution analysis, comparative evaluation of administration routes, and carcinogenicity testing. The therapeutic efficacy and safety of HPLCs were assessed in a fatal rat model of D-galactosamine (D-gal) and lipopolysaccharide (LPS)-induced ALF. In addition, the HPLCs underwent quality evaluation by the National Institutes for Food and Drug Control (NIFDC), and an independent safety assessment was conducted.ResultsA high-efficiency system was established for the generation of qualified, clinical-grade HPLCs from hESCs under GMP-compliant conditions. The HPLCs exhibited multiple mature hepatocyte functions, including carbohydrate and lipid metabolism, hepatic synthetic and storage functions, inducible cytochrome P450 activity, albumin secretion, and urea production. The HPLCs met the certification standards of the NIFDC of China. Transplantation of HPLCs significantly improved survival in ALF rats, with survival rates of 72.4% following tail vein injection and 66.7% following intraperitoneal injection, compared with 6.67% in the control group. HPLC transplantation also promoted recovery of liver function, as reflected by improvements in biochemical and coagulation parameters. Preclinical safety evaluations confirmed the biosafety of HPLCs, with no evidence of acute toxicity or tumorigenicity. A Phase I clinical trial (ChiCTR2100052988) for the treatment of ALF and acute-on-chronic liver failure (ACLF) has been approved by the National Health Commission of the People's Republic of China (Filing Number: MR-43-21-014643) and has been initiated.ConclusionsA novel multistage, GMP-compliant process was developed for the effective and reproducible differentiation of hESCs into hepatocytes. The resulting HPLCs demonstrated robust hepatocyte functions, therapeutic efficacy in an ALF animal model, and favorable biosafety profiles. These findings support the clinical translation of HPLCs, with an ongoing Phase I clinical trial designed to evaluate their safety and feasibility in patients with ALF and ACLF.

The precise assembly of the sperm flagellum is essential for male fertility and has long been ascribed to kinesin-2-driven intraflagellar transport (IFT) of protein cargoes. However, during late spermiogenesis, when transcription activity is largely silenced, how the spatiotemporally regulated delivery of flagellar components remains poorly understood. Here, we systematically screened kinesin genes in asthenozoospermic patients and identified two homozygous deleterious KIF6 variants in unrelated men characterized by complete sperm immotility. Mouse models carrying the corresponding mutations recapitulated the human infertility phenotypes. Multi-omics analyses revealed that most testicular mRNAs remained largely unchanged in Kif6 M1/M1 mice, whereas proteins involved in axonemal organization and energy metabolism were markedly reduced. Mechanistically, KIF6 interacts with the RNA-binding proteins (RBPs) FMRP and FXR1 to assemble mRNP transport complexes that ferry transcripts encoding flagellar structural proteins (e.g., DNALI1) and metabolic enzymes (e.g., HK1). Impaired KIF6 function compromises mRNP trafficking to the developing flagellum, reducing flagellar transcript levels and ultimately causing decreased protein abundance and defective flagellar function. Collectively, we identify KIF6 as a key regulator of mRNA transport during spermiogenesis. It interacts with RBPs through a novel IFT-like pathway to deliver mRNAs essential for flagellar biogenesis, redefining the traditional protein-centric IFT paradigm.

PURPOSE:Infertility affects about 15% of couples globally, with genetic factors contributing significantly. Advances in genomic technologies have led to the discovery of genes like MEI1, which play a crucial role in meiosis. However, the population prevalence and pathological mechanisms of human MEI1 variants remains poorly defined. METHODS:To elucidate the contribution of MEI1 to spermatogenic failure, initial screening for biallelic mutations was conducted in 626 non-obstructive azoospermia (NOA) patients, followed by targeted screening for heterozygous variants in a validation cohort comprising 1,607 well-characterized idiopathic male infertility cases (626 NOA, 799 severe oligoasthenospermia, and 182 terato/asthenozoospermia). RESULTS:Here, we identify MEI1 as a prominent gene associated with non-obstructive azoospermia (NOA), with biallelic pathogenic variants in MEI1 were identified in 12 individuals (1.9%, 12/626). Furthermore, heterozygous MEI1 mutations were identified in 28 patients (0.17%, 28/1607) from the broader infertility screening cohort implicate MEI1 in a wider phenotypic spectrum, broadening its clinical relevance. Critically, micro-TESE uniformly showed no sperm retrieval in individuals carrying biallelic MEI1 mutations, underscoring the imperative for preemptive genetic screening to avoid unnecessary surgical interventions. Mechanistically, these mutations disrupted interactions with key meiotic proteins (ANKRD31, IHO1, REC114, MEI4) in co-immunoprecipitation assays. CONCLUSION:These results not only elucidate its essential function in meiosis and DSB formation but also support its potential as a molecular marker for non-invasive diagnosis.

Oligoasthenoteratozoospermia (OAT) is a prevalent phenotype among infertile males, yet its underlying genetic etiology remains largely undefined. In this study, we found Spem2 deficiency did not affect the development of spermatogenic cells or the eventual generation of elongated spermatids within the testis, but affected the normal formation of residual bodies during the late stage of spermiation, which led to incomplete cytoplasmic removal of spermatids, abnormal sperm release and caused OAT. Affected sperm exhibited severely bent heads, cytoplasmic remnants encasing the head, and multiple sperm gathered. We further found SPEM2 may be involved in maintaining spermiation and cell polarity through interactions with cell polar molecules VANGL2, PRICKLE3, and DVL3. In vitro experiments showed that the transmembrane region of SPEM2 was its key functional domain and the binding domain for interactions between SPEM2 and VANGL2. Importantly, Spem2-deficient sperm enabled successful fertilization via intracytoplasmic sperm injection. Three novel SPEM2 heterozygous pathogenic variants were identified in four OAT patients by the whole-exome sequencing. Our findings revealed key roles of SPEM2 in spermiation, explored the potential pathogenesis of male infertility caused by SPEM2 deficiency, and provided essential information for genetic and reproductive counseling for such patients.

Non-obstructive azoospermia (NOA) and cryptozoospermia are two significant conditions contributing to male infertility. However, the underlying genetic factors in most cases remain unknown. In our study, whole exome sequencing identified novel biallelic variants in TBC1 domain family member 8 (TBC1D8) in two patients. Patient P1 with NOA harbored c.890C>T (p.A297V) and c.2461G>A (p.V821I), and patient P2 with cryptozoospermia carried c.854C>T (p.P285L) and c.1912G>A (p.D638N). Bioinformatic analyses predicted that all identified TBC1D8 variants were likely pathogenic. Compared with a normal control, patient P1 showed reduced expression of TBC1D8 in testicular tissue. Subsequently, hematoxylin-eosin staining and immunofluorescence analysis of testicular sections showed defective acrosome formation and the absence of elongated spermatids in patient P1, resulting from abnormal autophagy. Additionally, intracytoplasmic sperm injection treatment was beneficial for patient P2 with cryptozoospermia. In conclusion, our results suggest that TBC1D8 is a potentially novel candidate gene for male infertility associated with NOA or cryptozoospermia in humans.

Calmodulin (CaM) plays a crucial role in sperm function. Studies have reported that proteins containing the IQ motif interact with CaM, subsequently engaging with downstream target proteins known as calmodulin-binding proteins (CaMBPs). However, no relevant reports have been published detailing which CaMBPs exist and the mechanisms by which they are regulated. In this study, we conducted quantitative proteomic and phosphoproteomic analysis for mouse testes from wild-type (WT) and Iqcn knockout (Iqcn-/-) mice. The results indicated that Iqcn deficiency substantially rewires the downstream phosphorylation signaling pathway while not causing equivalent changes at protein levels. Among the 577 differentially regulated phosphorylated sites, most of them (494/577) belong to CaMBPs. Gene ontology analysis of these differentially phosphorylated CaMBPs showed enrichment in male gamete generation, actin cytoskeleton organization, and microtubule cytoskeleton organization process, demonstrating that IQCN regulates sperm function by interacting with CaM, which in turn affects the phosphorylation level of CaMBPs. Further kinase-substrate network analysis and the inhibition assay showed that FGFR4 and SYK tyrosine kinases are important for sperm motility and progressive motility. In summary, this study reveals that the interaction between IQCN and CaM regulates the phosphorylation of downstream CaMBPs and is involved in the related processes of spermiogenesis and sperm function.

Early embryonic development is controlled by maternal factors originating from mature oocytes. The zygotic genome is activated from a transcriptionally quiescent state through a process called embryonic genome activation (EGA), which involves the depletion and clearance of maternal factors. However, the mechanism by which maternal factors regulate EGA and embryonic development, particularly in humans, remains elusive. In this study, using tri-pronuclear (3PN) embryos and human embryonic stem cells (hESCs), we demonstrated that the maternal transcription factor Orthodenticle Homeobox 2 (OTX2), a paired-like homeobox gene, promotes EGA in human pre-implantation embryos. Knockdown of OTX2 through Trim-Away technology blocked embryonic development and minor EGA gene expression. Overexpression of OTX2 (OTX2OE) in hESCs increased transcript products, primarily at the 2-cell embryo stage genes, including genes encoding methyltransferase of histone H3K4. OTX2OE increased the level of H3K4me3 and increased the open chromatin region that co-occurs with the H3K4me3 region at the 4-cell stage in hESCs. Based on these findings in hESCs, we further verified that OTX2 directly induced the expression of SETD1A by binding to its promoter, leading to increased H3K4me3 levels in both hESCs and 3PN embryos. These findings suggest that the maternal transcription factor OTX2 regulates EGA and early embryogenesis via epigenetic mechanisms.

Research question Preimplantation genetic testing for monogenic diseases (PGT-M) is based on mutation testing combined with linkage analysis; so, how should PGT-M for patients with facioscapulohumeral dystrophy type 1 (FSHD1) be conducted when direct mutation testing is unavailable and specific linked markers are lacking? Design Patients with a definite FSHD1 diagnosis who intended to prevent the transmission of the condition to their children were recruited in the Reproductive and Genetic Hospital of CITIC-Xiangya, China between May 2021 and December 2023. Familial genetic risk was evaluated using Bionano, 4q haplotyping and whole-exome sequencing. Linkage markers near the causative 4q35 D4Z4 repeats were identified using targeted sequencing and MicroSeq. Haplotype analysis was conducted to determine affected and unaffected embryos. Embryo diagnosis rate was evaluated. Prenatal diagnosis through amniocentesis was offered at second trimester. Results Six patients with FSHD1 were included. During the pre-examination process of PGT-M, a single nucleotide polymorphism 3.5 kb upstream of the affected region using MicroSeq was identified, which could reduce misdiagnosis caused by recombination. A 4q-haplotype fast-determination method was used for determining 4qA and 4qB haplotypes, which could serve as downstream linkage markers. During PGT-M process, 34 blastocysts were evaluated for six families, with a diagnosis rate of 100%. Prenatal diagnosis using Bionano showed a normal 4q35 genotype in four families. Four healthy babies were born after six embryo transfers for five women. Conclusions Selecting effective linkage markers for PGT-M in FSHD1-affected regions is challenging. Successful PGT-M for FSHD1 is reported, which is of great clinical significance for genetic counselling and reproductive intervention for this disease.