STUDY QUESTION Does mono- (1PN) and tri-pronuclear (3PN) fertilization recapitulate the morphokinetic changes of normal bi-pronuclear (2PN) fertilization? SUMMARY ANSWER Abnormal fertilization retraces the overall choreography of normal fertilization but reveals novel morphokinetic phenomena and raises scientifically and clinically relevant questions. WHAT IS KNOWN ALREADY ART has allowed the extracorporeal observation of early human development. Time-lapse technology (TLT) has revealed the complexity of the morphokinetic changes underpinning fertilization and the importance of this process for the genetic and cellular integrity of the embryo. Abnormal fertilization has remained neglected, despite its relevance to the physiology and pathology of early human development. STUDY DESIGN, SIZE, DURATION This retrospective study involved TLT observation of normally (2PN, N = 2517) and abnormally (1PN, N = 41; 3PN, N = 27) fertilized oocytes generated in ICSI cycles performed between October 2019 and December 2020. Oocyte retrieval was carried out after clomiphene citrate-based minimal ovarian stimulation. Oocytes of patients with different diagnoses of infertility were included in the analysis, while cases involving cryopreserved gametes or surgically retrieved sperm were excluded. PARTICIPANTS/MATERIALS, SETTING, METHODS The study included 1231 couples treated for diverse infertility causes. The fraction of male factor cases was substantial (36.1%). Microinjected oocytes were assessed by a combined TLT-culture system. Oocytes not suitable for TLT assessment, owing to an excess of residual corona cells or inadequate orientation for correct observation, were not analysed. Phenomena relevant to meiotic resumption, pronuclear dynamics, cytoplasmic/cortical modifications, cleavage patterns and embryo quality were annotated and compared between groups. MAIN RESULTS AND THE ROLE OF CHANCE Extrusion of the second polar body (PBII) was observed in almost all 2PN/1PN (99.9% and 100.0%, respectively) and in a vast majority of 3PN zygotes (92.1%). Rates of PBII fusion with the ooplasm were much higher in 1PN and 3PN zygotes (P < 0.0001 versus 2PN). The cytoplasmic wave was observed not only in 2PN and 3PN but also in 1PN zygotes (positivity rates of 99.8% and 100% and 82.9%, respectively; P < 0.0001). More rarely, 2PN and 1PN zygotes emitted a third polar body (PBIII). The average times of this event were comparable. The presence and position of the cytoplasmic halo were comparable among the three classes of zygotes. In the 1PN group, the single PN was maternally or paternally derived in 17 and 24 zygotes, respectively, while in the vast majority of 3PN zygotes (121/127) the supernumerary PN was of maternal origin. Average times of maternal PN appearance were comparable, while average times of paternal PN appearance were delayed in 3PN zygotes (P = 0.0127). Compared with the control group, the area of the maternal PN was larger in 1PN zygotes, but smaller in 3PN zygotes (P < 0.0001). The paternal PNs displayed the same trend (P < 0.0001), although such values were consistently smaller than maternal PNs. The area of the third PN in the 3PN group was on average more than 50% smaller than those of maternal and paternal PNs. In maternal PNs of 3PN zygotes, nucleolus precursor bodies (NPBs) aligned along the area of PN juxtaposition at a lower rate compared with the 2PN group. The rate of NPB alignment was ∼50% smaller in 1PN zygotes (P = 0.0001). In paternal PNs, the rates of NPB alignment were not statistically different among the three groups. Asynchronous PN breakdown was increased in 3PN compared with 2PN zygotes (P = 0.0026). In 1PN zygotes, a developmental delay was observed starting from the disappearance of the cytoplasmic halo, reaching 9 h at the time of the first cleavage (P < 0.0001). Higher rates of abnormal cleavage patterns and blastomere fragmentation (P < 0.0001) were observed in 1PN compared to 2N and 3PN zygotes. Cleavage progression was increasingly affected after abnormal fertilization, especially 1PN, finally resulting in blastocyst formation rates of 70.2%, 12.2% and 53.5% in 2PN, 1PN and 3PN embryos, respectively (P < 0.0001). Both maternal and paternal ages were higher in cases involving 3PN fertilization. LIMITATIONS, REASONS FOR CAUTION The study data were obtained from ICSI, but not standard IVF, treatments carried out in a single centre. The study findings therefore require independent verification. WIDER IMPLICATIONS OF THE FINDINGS This study reports the first detailed morphokinetic map of human abnormal fertilization. Collectively, this evidence prompts new scientific hypotheses and raises clinical questions relevant to the aetiology and the treatment of abnormal fertilization. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the participating institutions. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER N/A.

中文翻译：

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人类 1PN 和 3PN 受精卵概括了正常受精的所有形态动力学事件，但揭示了新的发育错误

研究问题 单核 (1PN) 和三原核 (3PN) 受精是否概括了正常双原核 (2PN) 受精的形态动力学变化？总结 答案 异常受精追溯了正常受精的整体编排，但揭示了新的形态动力学现象，并提出了科学和临床相关的问题。什么是已知的 艺术已经允许对早期人类发育进行体外观察。延时技术 (TLT) 揭示了支撑受精的形态动力学变化的复杂性，以及这一过程对胚胎遗传和细胞完整性的重要性。尽管异常受精与人类早期发育的生理学和病理学相关，但它仍然被忽视。研究设计，尺寸，持续时间 本回顾性研究涉及 TLT 观察在 2019 年 10 月至 2020 年 12 月期间进行的 ICSI 周期中产生的正常（2PN，N = 2517）和异常（1PN，N = 41；3PN，N = 27）受精卵母细胞。进行了卵母细胞检索以克罗米芬为基础的最小卵巢刺激后。分析包括具有不同不孕症诊断的患者的卵母细胞，而涉及冷冻配子或手术取出精子的病例被排除在外。参与者/材料、环境、方法 该研究包括 1231 对因各种不孕原因接受治疗的夫妇。男性因素病例的比例很大（36.1%）。通过联合 TLT 培养系统评估显微注射的卵母细胞。卵母细胞不适合 TLT 评估，由于残留的电晕细胞过多或方向不足以正确观察，因此未进行分析。对与减数分裂恢复、原核动力学、细胞质/皮质修饰、卵裂模式和胚胎质量相关的现象进行了注释和组间比较。主要结果和机会的作用 在几乎所有 2PN/1PN（分别为 99.9% 和 100.0%）和绝大多数 3PN 受精卵（92.1%）中都观察到了第二极体 (PBII) 的挤出。在 1PN 和 3PN 受精卵中，PBII 与卵质的融合率要高得多（与 2PN 相比，P < 0.0001）。不仅在 2PN 和 3PN，而且在 1PN 受精卵中也观察到细胞质波（阳性率分别为 99.8% 和 100% 和 82.9%；P < 0.0001）。更罕见的是，2PN 和 1PN 受精卵发出第三极体 (PBIII)。该事件的平均时间具有可比性。细胞质晕的存在和位置在三类受精卵中具有可比性。在 1PN 组中，单一 PN 分别来自 17 和 24 个受精卵的母系或父系，而在绝大多数 3PN 受精卵 (121/127) 中，额外的 PN 是母系来源的。母亲 PN 出现的平均时间相当，而 3PN 受精卵中父亲 PN 出现的平均时间延迟（P = 0.0127）。与对照组相比，1PN受精卵母体PN面积较大，3PN受精卵较小（P＜0.0001）。父亲的 PN 表现出相同的趋势 (P < 0.0001)，尽管这些值始终小于母亲的 PN。3PN 组中第三次 PN 的面积平均比母系和父系 PN 小 50% 以上。在 3PN 受精卵的母体 PN 中，与 2PN 组相比，核仁前体体 (NPB) 沿 PN 并列区域排列的速率较低。在 1PN 受精卵中，NPB 比对率小 50%（P = 0.0001）。在父系 PN 中，NPB 对齐率在三组之间没有统计学差异。与 2PN 受精卵相比，3PN 的异步 PN 分解增加（P = 0.0026）。在 1PN 受精卵中，从细胞质晕消失开始观察到发育延迟，在第一次分裂时达到 9 小时（P < 0.0001）。与 2N 和 3PN 受精卵相比，在 1PN 中观察到更高的异常切割模式和卵裂球碎裂率 (P < 0.0001)。异常受精后卵裂进展受到越来越大的影响，尤其是1PN，最终导致2PN、1PN和3PN胚胎的囊胚形成率分别为70.2%、12.2%和53.5%（P＜0.0001）。在涉及 3PN 受精的情况下，母亲和父亲的年龄都较高。限制、谨慎的原因 研究数据来自 ICSI，但不是标准的 IVF，在单个中心进行的治疗。因此，研究结果需要独立验证。研究结果的更广泛意义 本研究报告了人类异常受精的第一个详细形态动力学图。总的来说，这一证据提出了新的科学假设，并提出了与病因学和异常受精治疗相关的临床问题。研究资金/竞争兴趣 本研究得到了参与机构的支持。作者没有需要声明的利益冲突。试用注册号 不适用。