Premature ovarian insufficiency (POI) is characterized by ovarian dysfunction that develops from diminished ovarian reserve (DOR). The exact aetiology of POI remains poorly understood. This study aims to elucidate the role of CKAP5 in the regulation of ovarian function and fertility. Bulk RNA sequencing of granulosa cells was conducted in the control group and in the patients with DOR to screen for candidate genes, which were further validated by gene burden analysis in a next-generation sequencing cohort of POI and control individuals. Additionally, ovarian reserve was evaluated in heterozygous Ckap5 knockout mice, alongside the ovarian and oocyte single-cell transcriptome analysis. The regulatory mechanism of CKAP5 was studied through in vivo and in vitro experiments. CKAP5 was identified as a key hub gene associated with ovarian ageing. Heterozygous Ckap5 knockout mice exhibited a POI-like phenotype, characterized by a reduced primordial follicle pool and accelerated follicular atresia. CKAP5 promotes autophagy via ATG7 and simultaneously supports DNA damage repair through the ATM. Finally, a variant in CKAP5 (NM_0001008938.4, c.630 + 7_630 + 11delCAAAA) was identified in patients with POI, resulting in protein truncation and loss of function. CKAP5 deficiency induces premature ovarian insufficiency in both humans and mice. The National Key R&D Program of China (2017YFC1001100), the National Natural Science Foundation of China (81501248, 81471453 and 81801295), the Health Research Project of Hunan Provincial Health Commission (W20243018), the Science and Technology Innovation Program of Hunan Province (2021RC3031), the National Natural Science Foundation of Hunan Province (2022JJ30066), the Scientific Research Program of Hunan Provincial Health Commission (202205033471 and 21B0058), the Open Research Fund of Hunan Provincial Key Laboratory of Regional Hereditary Birth Defects Prevention and Control (HPKL2023013).

Introduction: Asthenoteratozoospermia is one of the most common causes of male infertility. Several genes have been identified as genetic causative factors, but there is a considerable genetic heterogeneity underlying asthenoteratozoospermia. In this study, we performed a genetic analysis of two brothers from a consanguineous Uighur family in China to identify gene mutations causative for asthenoteratozoospermia-related male infertility.Methods: Two related patients with asthenoteratozoospermia from a large consanguineous family were sequenced by whole-exome sequencing and Sanger sequencing to identify disease-causing genes. Scanning and transmission electron microscopy analysis revealed ultrastructural abnormalities of spermatozoa. Quantitative real-time PCR (qRT-PCR) analysis and immunofluorescence (IF) analysis were used to assess the expression of the mutant messenger RNA (mRNA) and protein.Results: A novel homozygous frameshift mutation (c.2823dupT, p.Val942Cysfs*21) in DNAH6 was identified in both affected individuals and was predicted to be pathogenic. Papanicolaou staining and electron microscopy revealed multiple morphological and ultrastructural abnormalities of affected spermatozoa. qRT-PCR and IF analysis showed abnormal expression of DNAH6 in affected sperm, probably due to premature termination code and decay of abnormal 3′ untranslated region (UTR) region of mRNA. Furthermore, intracytoplasmic sperm injection could achieve successful fertilization in infertile men with DNAH6 mutations.Discussion: The novel frameshift mutation identified in DNAH6 may contribute to asthenoteratozoospermia. These findings expand the spectrum of genetic mutations and phenotypes associated with asthenoteratozoospermia and may be useful for genetic and reproductive counseling in male infertility.

Stroke is a leading risk factor for disability and death. Necroptosis is involved in stroke pathogenesis. However, the molecular mechanisms underlying necroptosis in stroke remain unclear. The mammalian target of rapamycin complex 1 (mTORC1) modulates necroptosis in the gut epithelium. Eukaryotic translation initiation factor 4E (eIF4E)‐binding protein‐1 (4EPB1) is one of the main downstream molecules of mTORC1. This study addresses the role of the 4EBP1–eIF4E pathway in necroptosis. The 4EBP1–eIF4E pathway was found to be activated in both necroptotic HT‐22 and mouse middle cerebral artery occlusion (MCAO) models. Functionally, 4EBP1 overexpression, eIF4E knockdown, and eIF4E inhibition suppressed necroptosis, respectively. Furthermore, a positive feedback circuit was observed between the 4EBP1–eIF4E and receptor‐interacting protein‐3 (RIP3)–mixed lineage kinase domain‐like protein (MLKL) pathways, in which RIP3–MLKL activates the 4EBP1–eIF4E pathway by degrading 4EBP1 and activating eIF4E. This in turn enhanced RIP3–MLKL pathway activation. The eIF4E activation derived from this loop may stimulate cytokine production, which is a key factor associated with necroptosis. Finally, using a mouse MCAO model, the application of eIF4E, RIP3, and MLKL inhibitors was found to have a regulatory mechanism similar to that in the in vitro study, reducing the infarct volume and improving neurological function in MCAO mice.

Introduction: Human zona pellucida (ZP) plays an important role in reproductive process. Several rare mutations in the encoding genes (ZP1, ZP2, and ZP3) have been demonstrated to cause women infertility. Mutations in ZP2 have been reported to cause ZP defects or empty follicle syndrome. We aimed to identify pathogenic variants in an infertile woman with a thin zona pellucida (ZP) phenotype and investigated the effect of ZP defects on oocyte gene transcription.Methods: We performed whole-exome sequencing and Sanger sequencing of genes were performed for infertilite patients characterized by fertilization failure in routine in vitro fertilization (IVF). Immunofluorescence (IF) and intracytoplasmic sperm injection (ICSI) were used in the mutant oocytes. Single-cell RNA sequencing was used to investigate transcriptomes of the gene-edited (Zp2^mut/mut) rat model. Biological function enrichment analysis, quantitative real-time PCR (qRT-PCR), and IF were performed.Results: We identified a novel homozygous nonsense mutation of ZP2 (c.1924C > T, p.Arg642X) in a patient with non-consanguineous married parents. All oocytes showed a thin or no ZP under a light microscope and were fertilized after ICSI. The patient successfully conceived by receiving the only two embryos that developed to the blastocyst stage. The immunofluorescence staining showed an apparently abnormal form of the stopped oocytes. We further demonstrated a total of 374 differentially expressed genes (DEGs) in the transcriptome profiles of Zp2^mut/mut rats oocytes and highlighted the signal communication between oocytes and granulosa cells. The pathway enrichment results of DEGs showed that they were enriched in multiple signaling pathways, especially the transforming growth factor-β (TGF-β) signaling pathway in oocyte development. qRT-PCR, IF, and phosphorylation analysis showed significantly downregulated expressions of Acvr2b, Smad2, p38MAPK, and Bcl2 and increased cleaved-caspase 3 protein expression.Discussion: Our findings expanded the known mutational spectrum of ZP2 associated with thin ZP and natural fertilization failure. Disruption of the integrity of the ZP impaired the TGF-β signaling pathway between oocytes and surrounding granulosa cells, leading to increased apoptosis and decreased developmental potential of oocytes.

Background: Paroxysmal kinesigenic dyskinesia (PKD) is the most prevalent kind type of paroxysmal Dyskinesia, characterized by recurrent and transient episodes of involuntary movements. Most PKD cases were attributed to the proline‐rich transmembrane protein 2 (PRRT2) gene, in which the c.649 region is a hotspot for known mutations. Even though some patients with PKD have been genetically diagnosed using whole‐exome sequencing (WES) and Sanger sequencing, there are still cases of missed diagnoses due to the limitations of sequencing technology and analytic methods on throughput.Methods: Patients meeting the diagnosis criteria of PKD with negative results of PRRT2‐Sanger sequencing and WES were included in this study. Mutation screening and targeted high‐throughput sequencing were performed to analyze and verify the sequencing results of the potential mutations.Results: Six patients with PKD with high mutation ratios of c.649dupC were screened using our targeted high‐throughput sequencing from 26 PKD patients with negative results of PRRT2‐Sanger sequencing and WES (frequency = 23.1%), which compensated for the comparatively shallow sequencing depth and statistical flaws in this region. Compared with the local normal population and other patients with PKD, the mutation ratios of c.649dupC of these six patients with PKD were much higher and also had truncated protein structures and differentially altered mRNA expression.Conclusion: Based on the above studies, we emphasize the routine targeted high‐throughput sequencing of the c.649 site in the PRRT2 gene in so‐called genetic‐testing‐negative patients with PKD, and manually calculate the deletion and duplication mutations depth and ratios to lower the rate of clinical misdiagnosis.