Reproductive and Genetic Hospital of CITIC-XIANGYA, Changsha, China.
Institute of Reproductive and Stem Cell Engineering, Basic Medicine College, Central South University, Changsha, China.
J Assist Reprod Genet. 2017 Sep;34(9):1189-1197. doi: 10.1007/s10815-017-0972-9. Epub 2017 Jun 22.
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).
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!
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.
We identified several candidate genes affecting pronucleus formation as a new cause of infertility.
本研究旨在探讨一位患者在体外受精（IVF）和胞浆内单精子注射（ICSI）后卵裂期胚胎反复出现多核（MPN）的原因。
这是一项病例研究。一位不明原因原发性不孕患者，在 IVF 和 ICSI 周期后出现反复 MPN 卵裂期胚胎。对原核形成进行时差监测。通过荧光原位杂交（FISH）分析 MPN 卵裂期胚胎。采用单细胞 RNA-seq 分析鉴定患者卵母细胞和胚胎的基因表达谱，并与正常受精的卵母细胞和胚胎（患者，n=1；供体，n=4）的数据进行比较。通过 Amazonia! 选择具有差异表达的卵母细胞特异性基因。
从时差分析中，我们观察到在第二次极体排出部位附近形成了多个微核。这些微核迁移、扩大并与雄性原核并列，导致多核。这些 MPN 卵裂期胚胎均不能发育至囊胚阶段，FISH 分析显示停滞胚胎的染色体组成混乱。RNA-seq 分析显示患者和供体卵母细胞和胚胎之间有 113 个差异表达基因（DEGs）。此外，113 个 DEGs 中有 25 个在卵母细胞和早期胚胎中特异或高表达。从 25 个 DEGs 中，进一步通过实时 PCR 验证了三个参与减数分裂和染色体分离过程的基因 DYNC2LI1、NEK2 和 CCNH。
我们确定了几个候选基因，这些基因可能影响原核形成，是导致不孕的新原因。
