Meiotic spindle assembly ensures proper chromosome segregation in oocytes. However, the mechanisms behind spindle assembly in human oocytes remain largely unknown. We used three-dimensional high-resolution imaging of more than 2000 human oocytes to identify a structure that we named the human oocyte microtubule organizing center (huoMTOC). The proteins TACC3, CCP110, CKAP5, and DISC1 were found to be essential components of the huoMTOC. The huoMTOC arises beneath the oocyte cortex and migrates adjacent to the nuclear envelope before nuclear envelope breakdown (NEBD). After NEBD, the huoMTOC fragments and relocates on the kinetochores to initiate microtubule nucleation and spindle assembly. Disrupting the huoMTOC led to spindle assembly defects and oocyte maturation arrest. These results reveal a physiological mechanism of huoMTOC-regulated spindle assembly in human oocytes.

Spindle bipolarization, the process of a microtubule mass transforming into a bipolar spindle, is a prerequisite for accurate chromosome segregation. In contrast to mitotic cells, the process and mechanism of spindle bipolarization in human oocytes remains unclear. Using high-resolution imaging in more than 1800 human oocytes, we revealed a typical state of multipolar intermediates that form during spindle bipolarization and elucidated the mechanism underlying this process. We found that the minor poles formed in multiple kinetochore clusters contribute to the generation of multipolar intermediates. We further determined the essential roles of HAUS6, KIF11, and KIF18A in spindle bipolarization and identified mutations in these genes in infertile patients characterized by oocyte or embryo defects. These results provide insights into the physiological and pathological mechanisms of spindle bipolarization in human oocytes.

Mitochondrial base editing with DddA‐derived cytosine base editor (DdCBE) is limited in the accessible target sequences and modest activity. Here, the optimized DdCBE tools is presented with improved editing activity and expanded C‐to‐T targeting scope by fusing DddA11 variant with different cytosine deaminases with single‐strand DNA activity. Compared to previous DdCBE based on DddA11 variant alone, fusion of the activation‐induced cytidine deaminase (AID) from Xenopus laevis not only permits cytosine editing of 5′‐GC‐3′ sequence, but also elevates editing efficiency at 5′‐TC‐3′, 5′‐CC‐3′, and 5′‐GC‐3′ targets by up to 25‐, 10‐, and 6‐fold, respectively. Furthermore, the A‐to‐G editing efficiency is significantly improved by fusing the evolved DddA6 variant with TALE‐linked deoxyadenosine deaminase (TALED). Notably, the authors introduce the reported high‐fidelity mutations in DddA and add nuclear export signal (NES) sequences in DdCBE and TALED to reduce off‐target editing in the nuclear and mitochondrial genome while improving on‐target editing efficiency in mitochondrial DNA (mtDNA). Finally, these engineered mitochondrial base editors are shown to be efficient in installing mtDNA mutations in human cells or mouse embryos for disease modeling. Collectively, the study shows broad implications for the basic study and therapeutic applications of optimized DdCBE and TALED.

STUDY QUESTION: Do novel per- and poly- fluoroalkyl substances (novel PFAS) have associations with premature ovarian insufficiency (POI)? SUMMARY ANSWER: Hexafluoropropylene oxide dimer acid (HFPO-DA), perfluorobutanoic acid (PFBA), perfluoropentanoic acid (PFPeA) and perfluoropentanesulfonic acid (PFPeS) are associated with an increased risk of POI, and the effect is worse whith exposure to mixtures. WHAT IS KNOWN ALREADY: As public health concerns following novel PFAS exposure are rising globally, there is a need to understand the exact association between ovel PFAS and various diseases. Epidemiologic studies suggest traditional PFAS exposures adversely affect women’s reproductive health, but the association between exposure to Novel PFAS and POI remains unclear. STUDY DESIGN, SIZE, DURATION: A retrospective research, including 371women, with (case group, n = 151) and without POI (control group, n = 220), were conducted between June 2023 and May 2024. PARTICIPANTS/MATERIALS, SETTING, METHODS: Thirteen types of Novel PFAS and basal concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2) and anti-Müllerian hormone (AMH) in plasma samples were measured in plasma samples collected during the early follicular phase (days 2–5) of a natural menstrual cycle. In addition, characteristics of participants were collected. Both adjusted logistic regression and Bayesian Kernel Machine Regression (BKMR) were used to evaluate associations between Novel PFAS (alone or as a mixture) and POI. Effect modification by age was also investigated. MAIN RESULTS AND THE ROLE OF CHANCE: The concentrations of HFPO-DA, PFBA, PFPeA and PFPeS in the case group were significantly higher than in the reference group. The adjusted logistic regression models demonstrated a positive associations between plasma concentrations of HFPO-DA, PFBA, PFPeA and PFPeS with the risk of POI [ORadj = 2.89 (95%CI: 1.84–4.53), 1.54 (95%CI: 1.17–2.02), 3.12 (95%CI: 2.20–4.43) and 2.07 (95%CI: 1.31–3.27), respectively, per 2.7-fold increase in Novel PFAS concentrations]. High concentrations of Novel PFAS showed a negative correlation with AMH and antral follicle count (AFC), but a positive correlation with FSH. After controlling for other covariates, HFPO-DA, PFBA, PFBS, PFPeA and PFPeS were the major contributors based on the BKMR models. LIMITATIONS, REASONS FOR CAUTION: False positives cannot be ruled out. Therefore, experiments on PFBA, PFPeA, PFPeS and HFPO-DA in vivo also need to be conducted in animal models. WIDER IMPLICATIONS OF THE FINDINGS: Our study is the first to discover the impact of Novel PFAS on the incidence of POI, with aninvestigation of indicators such as AMH, FSH, and AFC. Considering increasingly severe environmental pollution, our research results provides a broader understanding of the impact of environmental endocrine disruptors on ovarian function, and suggests that women of reproductive age should reduce their exposure to Novel PFAS. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Key Research and Development Project of China (2022YFC2703002), National Natural Science Foundation of China (U24A20658, 82371726), Innovative Research Team of High-Level Local Universities in Shanghai (SHSMU-ZDCX20212200), Shanghai Hospital Development Center Foundation (SHDC22022303, SHDC22022201) and Key project of Medical and Industrial intersection of Shanghai Jiao Tong University (YG2023ZD27), as well as Reproductive Medicine Research Project of the Chinese Red Cross Foundation (HSZH2024GFYZQ) and Open Fund Project of Shanghai Key Laboratory of Embryogenic Diseases (shelab2023ZD02). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. TRIAL REGISTRATION NUMBER: N/A