STUDY QUESTION Do biallelic deleterious variants of Calreticulin 3 (CALR3) cause fertilization failure (FF), resulting in male infertility in humans? SUMMARY ANSWER Biallelic mutations in CALR3 were identified in two infertile men from unrelated families and were shown to cause FF associated with failed sperm-zona pellucida (ZP) binding. WHAT IS KNOWN ALREADY In male mice, the Calr3-knockout has been reported to cause male infertility and FF. However, the mechanism behind this remains unclear in humans. STUDY DESIGN, SIZE, DURATION Sequencing studies were conducted in a research hospital on samples from Han Chinese families with primary infertility and sperm head deformations to identify the underlying genetic causes. PARTICIPANTS/MATERIALS, SETTING, METHODS Data from two infertile probands characterized by sperm head deformation were collected through in silico analysis. Sperm cells from the probands were characterized using light and electron microscopy and used to verify the pathogenicity of genetic factors through functional assays. Subzonal insemination (SUZI) and IVF assays were performed to determine the exact pathogenesis of FF. ICSI were administered to overcome CALR3-affected male infertility. MAIN RESULTS AND THE ROLE OF CHANCE Novel biallelic deleterious mutations in CALR3 were identified in two infertile men from unrelated families. We found one homozygous frameshift CALR3 mutation (M1: c.17_27del, p.V6Gfs*34) and one compound heterozygous CALR3 mutation (M2: c.943A>G, p.N315D; M3: c.544T>C, p.Y182H). These mutations are rare in the general population and cause acrosomal ultrastructural defects in affected sperm. Furthermore, spermatozoa from patients harbouring the CALR3 mutations were unable to bind to the sperm-ZP or they disrupted gamete fusion or prevented oocyte activation. Molecular assays have revealed that CALR3 is crucial for the maturation of the ZP binding protein in humans. Notably, the successful fertilization via SUZI and ICSI attempts for two patients, as well as the normal expression of PLCζ in the mutant sperm, suggests that ICSI is an optimal treatment for CALR3-deficient FF. LIMITATIONS, REASONS FOR CAUTION The results are based on sperm-related findings from two patients. Further studies are required to gain insight into the developmental stage and function of CALR3 in human testis. WIDER IMPLICATIONS OF THE FINDINGS Our findings highlight the underlying risk of FF associated with sperm defects and provide a valuable reference for personalized genetic counselling and clinical treatment of these patients. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the National Key R&D Program of China (2021YFC2700901), Hefei Comprehensive National Science Center Medical-Industrial Integration Medical Equipment Innovation Research Platform Project (4801001202), the National Natural Science Foundation of China (82201803, 82371621, 82271639), Foundation of the Education Department of Anhui Province (gxgwfx2022007), Key Project of Natural Science Research of Anhui Educational Committee (2023AH053287), and the Clinical Medical Research Transformation Project of Anhui Province (202204295107020037). The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A.

中文翻译：

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CALR3 缺陷破坏人类精子-透明带结合：对男性因子受精失败和相关临床治疗方法的新见解。


研究问题钙网蛋白 3 （CALR3） 的双等位基因有害变体会导致受精失败 （FF），从而导致人类男性不育吗？摘要答案 在来自无关家庭的两名不育男性中发现了 CALR3 的双等位基因突变，并被证明会导致与精子透明带 （ZP） 结合失败相关的 FF。已知的在雄性小鼠中，据报道 Calr3 敲除会导致雄性不育和 FF。然而，这在人类中背后的机制仍不清楚。研究设计、规模、持续时间 测序研究在一家研究医院对患有原发性不孕症和精子头部变形的汉族家庭的样本进行，以确定潜在的遗传原因。参与者/材料、设置、方法 通过计算机分析收集来自两种以精子头部变形为特征的不育先证者的数据。使用光学和电子显微镜对先证者精子细胞进行表征，并用于通过功能测定验证遗传因子的致病性。进行亚带授精 （SUZI） 和 IVF 测定以确定 FF 的确切发病机制。施用 ICSI 以克服受 CALR3 影响的男性不育症。主要结果和机会的作用 在来自无关家庭的两名不育男性中发现了 CALR3 中的新型双等位基因有害突变。我们发现了一个纯合移码 CALR3 突变 （M1： c.17_27del， p.V6Gfs*34） 和一个复合杂合 CALR3 突变 （M2： c.943A>G， p.N315D;M3： c.544T>C， p.Y182H）.这些突变在普通人群中很少见，并导致受影响精子的顶体超微结构缺陷。 此外，携带 CALR3 突变的患者的精子无法与精子 ZP 结合，或者它们破坏了配子融合或阻止了卵母细胞活化。分子检测显示，CALR3 对于人类 ZP 结合蛋白的成熟至关重要。值得注意的是，两名患者通过 SUZI 和 ICSI 尝试成功受精，以及突变精子中 PLCζ 的正常表达，表明 ICSI 是 CALR3 缺陷型 FF 的最佳治疗方法。局限性，谨慎的原因 结果基于两名患者的精子相关发现。需要进一步的研究来深入了解 CALR3 在人类睾丸中的发育阶段和功能。研究结果的更广泛意义 我们的研究结果强调了与精子缺陷相关的 FF 的潜在风险，并为这些患者的个性化遗传咨询和临床治疗提供了有价值的参考。研究资金/利益争夺本研究得到了国家重点研发计划（2021YFC2700901）、合肥市综合性国家科学中心医工融合医疗器械创新研究平台项目（4801001202）、国家自然科学基金（82201803、82371621、82271639）、安徽省教育厅基金（gxgwfx2022007）、安徽省教育委员会自然科学研究重点项目（2023AH053287），安徽省临床医学研究转化项目（202204295107020037）。作者声明没有利益冲突。试验注册号 N/A。