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. 2024 Apr 23;2024(2):hoae023.
doi: 10.1093/hropen/hoae023. eCollection 2024.

The severity of meiotic aneuploidy is associated with altered morphokinetic variables of mouse oocyte maturation

Affiliations

The severity of meiotic aneuploidy is associated with altered morphokinetic variables of mouse oocyte maturation

Yiru Zhu et al. Hum Reprod Open. .

Abstract

Study question: Is there an association between morphokinetic variables of meiotic maturation and the severity of aneuploidy following in vitro maturation (IVM) in the mouse?

Summary answer: The severity of meiotic aneuploidy correlates with an extended time to first polar body extrusion (tPB1) and duration of meiosis I (dMI).

What is known already: 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.

Study design size duration: 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).

Participants/materials setting 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.

Main results and the role of chance: 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).

Large scale data: N/A.

Limitations reasons for caution: 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.

Wider implications of the findings: 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.

Study funding/competing interests: 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.

Keywords: aneuploidy; chromosomal abnormalities; in vitro maturation; meiosis; oocyte quality; time-lapse microscopy.

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Conflict of interest statement

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.The authors have no conflict of interest to disclose.

Figures

Figure 1.
Figure 1.
Overview of using whole genome amplification (WGA) and next generation sequencing (NGS) for conducting sex concordance and reciprocal aneuploidy analysis. (A) Commercially available mouse blastomeres and matched meiosis II (MII) oocyte/polar body pairs were used for platform validation. The oocyte and its matched polar body were separated following removal of the zona pellucida following treatment with Acidic Tyrode’s solution. The number of X and Y chromosomes were compared among different blastomeres of the same mouse embryo or with matched oocyte/polar body pairs following WGA and NGS. (B) IVM was performed on immature oocytes at GV stage in the EmbryoScope+™ with either DMSO (control), 25 or 50 nM of Nocodazole. After IVM, morphokinetic variables were determined and reciprocal aneuploidy analysis was conducted on MII oocytes to determine the changes in chromatid copy number. WGA, whole-genome amplification; NGS, next-generation sequencing; GV, germinal vesicle; GVBD, germinal vesicle breakdown; PBE, polar body extrusion; MI, meiosis I.
Figure 2.
Figure 2.
The effect of Nocodazole on oocyte maturation and spindle morphology. (A) Representative EmbryoScope+™ images of denuded oocytes treated with 25 or 50 nM Nocodazole following IVM. (B) Graph of meiotic progression for denuded oocytes treated with DMSO (control) and Nocodazole following IVM. Grey and black dots denote the percentage of oocytes at the GVBD or PBE stage, respectively, per replicate. (C) Representative immunofluorescent images showcasing the morphologies of control or Nocodazole-treated denuded oocytes during IVM. Actin (red), α-tubulin (green), and DNA (blue) were detected by immunocytochemistry. Scale bar = 25 µm. (D) Quantification of varying degrees of spindle abnormalities observed across control and Nocodazole-treated oocytes. N = 11–23 oocytes per treatment group across two replicates. GVBD, germinal vesicle breakdown; PBE, polar body extrusion; ns, not significant.
Figure 3.
Figure 3.
Reciprocal aneuploidy analysis on matched MII oocyte and polar body pairs reveals nondisjunction (NDJ) of homologs and premature separation of sister chromatids (PSSC). Representative chromosome plots classifying the severity of reciprocal aneuploidy as (A) euploid, (B) 1–3 aneuploidy events, and (C) ≥4 reciprocal aneuploidy events. Chromosomes with abnormal copy numbers are labeled at the top of the bars. PB, polar body.
Figure 4.
Figure 4.
The effect of Nocodazole on ploidy status and morphokinetic variables of meiotic progression. (A) Representative EmbryoScope+™ images of the control and Nocodazole-treated oocytes during IVM. Germinal vesicle and timepoint of GVBD are indicated by arrows (black) and solid black squares, respectively. Timepoint of the first extruded polar body (asterisk) is indicated by the dotted squares. (B) Graph of the proportion of oocytes within each reciprocal aneuploidy categorization (euploid, 1–3, or ≥4 aneuploidy events). (C–E) Quantification of (C) tGVBD, (D) tPB1, and (E) dMI in oocytes treated with DMSO (control) or different concentrations of Nocodazole. N = 11 (control), 13 (25 nM), and 23 (50 nM) oocytes across two replicates. GVBD, germinal vesicle breakdown; tGVBD, time to GVBD; tPB1, time to extrusion of the first polar body; dMI, duration of meiosis I; ns, not significant. ****P < 0.0001.
Figure 5.
Figure 5.
The effect of ploidy status on morphokinetic variables of meiotic progression. (A) Representative EmbryoScope+™ images of oocytes with different ploidy status during IVM. Germinal vesicle and timepoint of GVBD are indicated by arrows (black) and solid black squares, respectively. Timepoint of the first extruded polar body (asterisk) is indicated by the dotted squares. (C–E) Quantification of (C) tGVBD, (D) tPB1, and (E) dMI in oocytes of different ploidy status. N = 27 (euploid), 9 (1–3 aneuploidy events), and 11 (≥4 aneuploidy events) oocytes across two replicates. GVBD, germinal vesicle breakdown; tGVBD, time to GVBD; tPB1, time to extrusion of the first polar body; dMI, duration of meiosis I; ns, not significant. ****P < 0.0001.

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