目的:小鼠减数分裂成熟的形态动力学变量与体外成熟(IVM)后的非整倍体严重程度之间是否存在关联?
结论:减数分裂非整倍体的严重程度与第一极体挤压时间(tPpot)和减数分裂I期(dMI)的持续时间相关。
背景:使用延时技术测量的形态动力学变量允许在临床辅助生殖技术(ART)中对植入前胚胎发育进行非侵入性评估。我们最近应用这项技术来监测小鼠配子IVM期间减数分裂的进展。尚未在特定染色体的分辨率下系统地检查减数分裂进程的形态动力学变量与所得卵的非整倍性之间是否存在关系。下一代测序(NGS)是用于确定非整倍性状态的强大临床工具,并已在小鼠胚泡和卵母细胞中进行了反向翻译。因此,我们利用延时成像和NGS技术来确定减数分裂进展的形态动力学与卵非整倍体之间的关系.
■从来自过度刺激的CD-1小鼠的大腔卵泡中收集卵丘-卵母细胞复合物。卵丘细胞被移除,在不存在或存在两种剂量的Nocodazole(25或50nM)的情况下进行自发性IVM,以在卵母细胞减数分裂期间诱导一系列纺锤体异常和染色体分离错误。然后对所得的卵子进行全面的染色体筛查,和形态动力学变量和倍性状态在实验组(对照组,n=11;25nMNocodazole,n=13;50nMNocodazole,n=23)。
方法:我们使用延时显微镜监测小鼠卵母细胞的IVM16小时,和生发囊泡破裂时间(tGVBD),分析了tPB1和dMI。在IVM之后,通过基于NGS的非整倍性植入前遗传学检测(PGT-A)分析,对卵子及其匹配的第一极体进行了全面的染色体筛查.进行生物信息学分析以将读段与小鼠基因组比对,并确定非整倍性的基于拷贝数的预测。使用每个极体-卵对的一致性(倒数误差)来验证结果。倍性状态被归类为整倍体,1-3个染色体分离错误,或≥4个染色体分离错误。此外,区分了由于姐妹染色单体(PSSC)与非分离(NDJ)过早分离而导致的非整倍体。
结果:我们应用并验证了最新的NGS技术,以在染色体特异性水平上筛选单个小鼠卵和匹配极体的非整倍体。通过在不同剂量的Nocodazole存在下进行IVM,我们诱导了一系列的非整倍性。在没有Nocodazole(0/11)的情况下,没有观察到非整倍性,而在25和50nMNocodazole存在下的IVM导致7.69%(1/13)和82.61%(19/23)的非整倍性发生率,分别。在非整倍体卵中,5%(1/20)是由于PSSC,65%(13/20)至NDJ,剩下的是两者的组合。倍性状态与tGVBD之间没有关系,但是与整倍体卵相比,具有相互非整倍体事件的卵中的tPB1和dMI均显着延长,这与非整倍体的严重程度成比例。具有≥4个非整倍体染色体的鸡蛋具有最长的tPB1和dMI(P<0.0001),而具有一到三个非整倍体染色体的卵表现出中等时间长度(P<0.0001)。
方法:不适用。
结论:我们在本研究中使用诺考达唑来破坏减数分裂纺锤体并诱导小鼠卵母细胞的非整倍体。减数分裂进程的形态动力学变量与非整倍性的严重程度之间的关联是否与诱导染色体分离错误的其他化合物发生仍有待研究。此外,与小鼠卵母细胞不同,人类IVM需要卵丘细胞的存在,这排除了减数分裂进展的形态动力学变量的可视化。因此,我们的研究可能具有有限的直接临床可译性.
结论:我们在小鼠卵中验证了NGS以染色体特异性分辨率检测非整倍体,这大大提高了小鼠模型的实用性。使用易于处理且经过验证的模型系统来表征减数分裂非整倍体,可以进一步详细研究可能影响非整倍性的分子机制和因素。减数分裂进展的形态动力学变量的延时分析可能是非整倍体严重程度的有用非侵入性预测指标。
背景:这项工作得到了比尔和梅林达·盖茨基金会(INV-003385)的支持。根据基金会的资助条件,知识共享署名4.0通用许可证已分配给作者接受的手稿版本,该版本可能来自此提交。作者没有利益冲突要披露。
OBJECTIVE: Is there an association between morphokinetic variables of meiotic maturation and the severity of aneuploidy following in vitro maturation (IVM) in the mouse?
CONCLUSIONS: The severity of meiotic aneuploidy correlates with an extended time to first polar body extrusion (tPB1) and duration of meiosis I (dMI).
BACKGROUND: Morphokinetic variables measured using time-lapse technology allow for the non-invasive evaluation of preimplantation embryo development within clinical assisted reproductive technology (ART). We recently applied this technology to monitor meiotic progression during IVM of mouse gametes. Whether there is a relationship between morphokinetic variables of meiotic progression and aneuploidy in the resulting egg has not been systematically examined at the resolution of specific chromosomes. Next-generation sequencing (NGS) is a robust clinical tool for determining aneuploidy status and has been reverse-translated in mouse blastocysts and oocytes. Therefore, we harnessed the technologies of time-lapse imaging and NGS to determine the relationship between the morphokinetics of meiotic progression and egg aneuploidy.
UNASSIGNED: Cumulus-oocyte complexes were collected from large antral follicles from hyperstimulated CD-1 mice. Cumulus cells were removed, and spontaneous IVM was performed in the absence or presence of two doses of Nocodazole (25 or 50 nM) to induce a spectrum of spindle abnormalities and chromosome segregation errors during oocyte meiosis. Comprehensive chromosome screening was then performed in the resulting eggs, and morphokinetic variables and ploidy status were compared across experimental groups (control, n = 11; 25 nM Nocodazole, n = 13; 50 nM Nocodazole, n = 23).
METHODS: We monitored IVM in mouse oocytes using time-lapse microscopy for 16 h, and time to germinal vesicle breakdown (tGVBD), tPB1, and dMI were analyzed. Following IVM, comprehensive chromosome screening was performed on the eggs and their matched first polar bodies via adaptation of an NGS-based preimplantation genetic testing for aneuploidy (PGT-A) assay. Bioinformatics analysis was performed to align reads to the mouse genome and determine copy number-based predictions of aneuploidy. The concordance of each polar body-egg pair (reciprocal errors) was used to validate the results. Ploidy status was categorized as euploid, 1-3 chromosomal segregation errors, or ≥4 chromosomal segregation errors. Additionally, aneuploidy due to premature separation of sister chromatids (PSSC) versus non-disjunction (NDJ) was distinguished.
RESULTS: We applied and validated state-of-the-art NGS technology to screen aneuploidy in individual mouse eggs and matched polar bodies at the chromosome-specific level. By performing IVM in the presence of different doses of Nocodazole, we induced a range of aneuploidy. No aneuploidy was observed in the absence of Nocodazole (0/11), whereas IVM in the presence of 25 and 50 nM Nocodazole resulted in an aneuploidy incidence of 7.69% (1/13) and 82.61% (19/23), respectively. Of the aneuploid eggs, 5% (1/20) was due to PSSC, 65% (13/20) to NDJ, and the remainder to a combination of both. There was no relationship between ploidy status and tGVBD, but tPB1 and the dMI were both significantly prolonged in eggs with reciprocal aneuploidy events compared to the euploid eggs, and this scaled with the severity of aneuploidy. Eggs with ≥4 aneuploid chromosomes had the longest tPB1 and dMI (P < 0.0001), whereas eggs with one to three aneuploid chromosomes exhibited intermediate lengths of time (P < 0.0001).
METHODS: N/A.
CONCLUSIONS: We used Nocodazole in this study to disrupt the meiotic spindle and induce aneuploidy in mouse oocytes. Whether the association between morphokinetic variables of meiotic progression and the severity of aneuploidy occurs with other compounds that induce chromosome segregation errors remain to be investigated. In addition, unlike mouse oocytes, human IVM requires the presence of cumulus cells, which precludes visualization of morphokinetic variables of meiotic progression. Thus, our study may have limited direct clinical translatability.
CONCLUSIONS: We validated NGS in mouse eggs to detect aneuploidy at a chromosome-specific resolution which greatly improves the utility of the mouse model. With a tractable and validated model system for characterizing meiotic aneuploidy, investigations into the molecular mechanisms and factors which may influence aneuploidy can be further elaborated. Time-lapse analyses of morphokinetic variables of meiotic progression may be a useful non-invasive predictor of aneuploidy severity.
BACKGROUND: This work was supported by the Bill & Melinda Gates Foundation (INV-003385). Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission. The authors have no conflict of interest to disclose.