Time-lapse observation and transcriptome analysis of a case with repeated multiple pronuclei after IVF/ICSI
Time-lapse observation and transcriptome analysis of a case with repeated multiple pronuclei after IVF/ICSI
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IVF/ICSI后重复多原核病例的延时观察及转录组分析
DOI：
10.1007/s10815-017-0972-9
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发表时间：
2017-09-01
影响因子：
3.1
通讯作者：
Lin, G.
中科院分区：
文献类型：
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作者：
Dai, J.;Leng, L. Z.;Lin, G.
目的本研究的目的是探讨体外受精（IVF）和卵胞浆内单精子注射（ICSI）后患者受精卵反复形成多原核（MPN）的原因。方法这是一个案例研究。一名患者患有不明原因的原发性不孕症，在 IVF 和 ICSI 周期后复发总 MPN 合子。对原核形成进行延时监测。通过荧光原位杂交 (FISH) 分析 MPN 受精卵发育的胚胎。使用单细胞 RNA-seq 分析来识别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捐赠者（患者，n=1；捐赠者，n=4）的卵母细胞和受精卵的数据进行比较。亚马逊流域选择了具有差异表达的卵母细胞特异性基因！结果通过延时分析，我们观察到第二极体挤出部位附近形成了多个微核。这些微核迁移、扩张并与雄性原核并置，形成多原核。这些 MPN 受精卵均无法发育至囊胚阶段，FISH 分析显示，受阻胚胎中存在混乱的染色体补体。 RNA-seq 分析显示患者与供体卵母细胞和受精卵之间存在 113 个差异表达基因 (DEG)。此外，113 个 DEG 中有 25 个在卵母细胞和早期胚胎中是独特的或高度表达的。通过实时PCR进一步验证了25个DEG中参与减数分裂和染色体分离过程的三个基因DYNC2LI1、NEK2和CCNH。结论我们鉴定了几个影响原核形成的候选基因作为不孕的新原因。
PurposeThe purpose of this study was to investigate the cause of repeated multipronucleus (MPN) formation in zygotes in a patient after both in vitrofertilization(IVF) and intracytoplasmic sperm injection (ICSI).MethodThis 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! database.ResultsFrom 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, andCCNH, which are involved in meiosis and the chromosome separation process, were further validated by real-time PCR.ConclusionWe identified several candidate genes affecting pronucleus formation as a new cause of infertility.