J Dai1,2, L Z Leng2, C F Lu1,2,3, F Gong1,2,3, S P Zhang1,2, W Zheng1, G X Lu1,3,4, G Lin5,6,7,8. 1. Reproductive and Genetic Hospital of CITIC-XIANGYA, Changsha, China. 2. Institute of Reproductive and Stem Cell Engineering, Basic Medicine College, Central South University, Changsha, China. 3. National Engineering and Research Center of Human Stem Cell, Changsha, China. 4. Key Laboratory of Reproductive and Stem Cell Engineering, Ministry of Health, Changsha, China. 5. Reproductive and Genetic Hospital of CITIC-XIANGYA, Changsha, China. linggf@hotmail.com. 6. Institute of Reproductive and Stem Cell Engineering, Basic Medicine College, Central South University, Changsha, China. linggf@hotmail.com. 7. National Engineering and Research Center of Human Stem Cell, Changsha, China. linggf@hotmail.com. 8. Key Laboratory of Reproductive and Stem Cell Engineering, Ministry of Health, Changsha, China. linggf@hotmail.com.
Abstract
PURPOSE: 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). METHOD: 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! 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. CONCLUSION: We identified several candidate genes affecting pronucleus formation as a new cause of infertility.
PURPOSE: 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). METHOD: 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! 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. CONCLUSION: We identified several candidate genes affecting pronucleus formation as a new cause of infertility.
Authors: T Adriaenssens; S Wathlet; I Segers; G Verheyen; A De Vos; J Van der Elst; W Coucke; P Devroey; J Smitz Journal: Hum Reprod Date: 2010-03-13 Impact factor: 6.918