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Abstract：
OBJECTIVE: The purpose of this study was to investigate the cause of repeated multipronucleus (MPN) formation in zygotes in a patient after both in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).
METHODS: This is a case study. A patient had unexplained primary infertility with recurring total MPN zygotes after IVF and ICSI cycles. Time-lapse monitoring of pronucleus formation was carried out. Embryos developed from MPN zygotes were analyzed by fluorescence in situ hybridization (FISH). Single-cell RNA-seq analysis was used to identify gene expression profiles of the patient\'s oocyte and zygote, and these were compared to the data from oocytes and zygotes from donors with normal fertilization (patient, n = 1; donors, n = 4). Oocyte-specific genes with differential expression were selected by the Amazonia!
METHODS:
RESULTS: From time-lapse analysis, we observed the formation of multiple micronuclei near the site of the second polar body extrusion. These micronuclei migrated, expanded, and juxtaposed with the male pronucleus leading to a multipronucleus. None of these MPN zygotes could develop to the blastocyst stage, and FISH analysis revealed a chaotic chromosomal complement in the arrested embryos. RNA-seq analysis showed 113 differentially expressed genes (DEGs) between the patient and the donor oocytes and zygotes. Moreover, 25 of the 113 DEGs were unique or highly expressed in oocytes and early embryos. From 25 DEGs, three genes, DYNC2LI1, NEK2, and CCNH, which are involved in meiosis and the chromosome separation process, were further validated by real-time PCR.
CONCLUSIONS: We identified several candidate genes affecting pronucleus formation as a new cause of infertility.
METHODS: This is a case study. A patient had unexplained primary infertility with recurring total MPN zygotes after IVF and ICSI cycles. Time-lapse monitoring of pronucleus formation was carried out. Embryos developed from MPN zygotes were analyzed by fluorescence in situ hybridization (FISH). Single-cell RNA-seq analysis was used to identify gene expression profiles of the patient\'s oocyte and zygote, and these were compared to the data from oocytes and zygotes from donors with normal fertilization (patient, n = 1; donors, n = 4). Oocyte-specific genes with differential expression were selected by the Amazonia!
METHODS:
RESULTS: From time-lapse analysis, we observed the formation of multiple micronuclei near the site of the second polar body extrusion. These micronuclei migrated, expanded, and juxtaposed with the male pronucleus leading to a multipronucleus. None of these MPN zygotes could develop to the blastocyst stage, and FISH analysis revealed a chaotic chromosomal complement in the arrested embryos. RNA-seq analysis showed 113 differentially expressed genes (DEGs) between the patient and the donor oocytes and zygotes. Moreover, 25 of the 113 DEGs were unique or highly expressed in oocytes and early embryos. From 25 DEGs, three genes, DYNC2LI1, NEK2, and CCNH, which are involved in meiosis and the chromosome separation process, were further validated by real-time PCR.
CONCLUSIONS: We identified several candidate genes affecting pronucleus formation as a new cause of infertility.
摘要：
目的：本研究的目的是探讨体外受精（IVF）和胞浆内单精子注射（ICSI）后患者受精卵中重复多核（MPN）形成的原因。
方法：这是一个案例研究。一名患者在IVF和ICSI周期后患有无法解释的原发性不孕症，并反复出现总MPN受精卵。进行了原核形成的延时监测。通过荧光原位杂交（FISH）分析从MPN受精卵发育的胚胎。单细胞RNA-seq分析用于鉴定患者卵母细胞和受精卵的基因表达谱，并将这些数据与正常受精供体的卵母细胞和受精卵的数据进行比较（患者，n=1；捐赠者，n=4)。通过亚马逊选择具有差异表达的卵母细胞特异性基因！
方法：
结果：从延时分析，我们观察到在第二极体挤压部位附近形成了多个微核。这些微核迁移，展开,并与男性原核并列，导致多核。这些MPN受精卵都不能发育到胚泡期，和FISH分析显示，在被捕的胚胎中出现了混乱的染色体补体。RNA-seq分析显示，患者与供体卵母细胞和受精卵之间有113个差异表达基因（DEG）。此外，113个DEGs中有25个在卵母细胞和早期胚胎中独特或高表达。从25度开始，三个基因,DYNC2LI1、NEK2和CCNH,它们参与减数分裂和染色体分离过程，通过实时PCR进一步验证。
结论：我们确定了几个影响原核形成的候选基因是不孕症的新原因。
方法：这是一个案例研究。一名患者在IVF和ICSI周期后患有无法解释的原发性不孕症，并反复出现总MPN受精卵。进行了原核形成的延时监测。通过荧光原位杂交（FISH）分析从MPN受精卵发育的胚胎。单细胞RNA-seq分析用于鉴定患者卵母细胞和受精卵的基因表达谱，并将这些数据与正常受精供体的卵母细胞和受精卵的数据进行比较（患者，n=1；捐赠者，n=4)。通过亚马逊选择具有差异表达的卵母细胞特异性基因！
方法：
结果：从延时分析，我们观察到在第二极体挤压部位附近形成了多个微核。这些微核迁移，展开,并与男性原核并列，导致多核。这些MPN受精卵都不能发育到胚泡期，和FISH分析显示，在被捕的胚胎中出现了混乱的染色体补体。RNA-seq分析显示，患者与供体卵母细胞和受精卵之间有113个差异表达基因（DEG）。此外，113个DEGs中有25个在卵母细胞和早期胚胎中独特或高表达。从25度开始，三个基因,DYNC2LI1、NEK2和CCNH,它们参与减数分裂和染色体分离过程，通过实时PCR进一步验证。
结论：我们确定了几个影响原核形成的候选基因是不孕症的新原因。
