Assessment of developmental potential of human single pronucleated zygotes derived from conventional in vitro fertilization
The aim of this study was to non-invasively validate the developmental potential of human single pronucleated (1PN) zygotes derived from conventional in vitro fertilization (c-IVF) at the zygote stage.
Fifty 1PN zygotes derived from 45 patients undergoing c-IVF were used. Immunohistochemistry and fluorescence live cell imaging were used to confirm normal chromosome segregation during the first mitosis. The usefulness of measuring pronuclear diameter was assessed on the basis of the presence or absence of a proper first cleavage and validated by subsequent development.
Although approximately 80% (15/19) of 1PN zygotes contained a diploid genome, immunohistochemistry revealed an unequal distribution of paternal and maternal genomes at the first mitosis. Fluorescence live imaging revealed that 73% (8/11) of 1PN zygotes formed a functional mitotic spindle at the first mitosis resulting from diploid genomes, with 25% (2/8) of these forming a tripolar spindle. 1PN zygotes in which the pronucleus disappeared and that subsequently underwent cleavage had a pronuclear diameter ≥ 32.2 μm. The selection of 1PN zygotes based on pronuclear diameter resulted in zygotes that all formed mitotic spindles with poles during cleavage. Furthermore, 63% (5/8) of these zygotes reached the blastocyst stage.
This study demonstrates the usefulness of a non-invasive assessment of 1PN zygotes derived from c-IVF as an indicator of developmental potential. Furthermore, diploid 1PN zygotes do not always exhibit normal chromosome segregation at the first mitosis. A pronuclear diameter ≥ 32.2 μm just before PN breakdown might be a useful criterion to assess 1PN zygotes that are capable of further development.
KeywordsSingle pronucleus (1PN) Mitotic spindle Chromosome segregation Fluorescence live cell imaging
Histone H3 trimethyl lysine 9
Microtubule associated protein 4
Pronucleus (plural: pronuclei)/pronuclear
Conventional in vitro fertilization
Intracytoplasmic sperm injection
Preimplantation genetic screening
The authors wish to thank Dr. Takeshi Matsui for assistance with the plasmid vector constructions. We are grateful to all the staff of the Mio Fertility Clinic for their assistance and encouragement.
Y.K. designed this research, performed experiments, analyzed the data, and wrote the manuscript. H.M. also analyzed the data and wrote the manuscript. K.Y and K.I were involved in the clinical zygote vitrification. Y.M. supervised this research.
Compliance with ethical standards
This study was approved by the Ethics Committee of JISART (Japanese Institution for Standardizing Assisted Reproductive Technology) and was registered with the Japan Society of Obstetrics and Gynecology.
Conflict of interest
The authors declare that they have no conflict of interest.
- 13.Cibelli JB, Kiessling AA, Cunniff K, Richards C, Lanza RP, West MD. Somatic cell nuclear transfer in humans: pronuclear and early embryonic development. J Regen Med. 2001;2:25–31.Google Scholar
- 16.Gardner DK, Schoolcraft WB. In vitro culture of human blastocysts. In: Jansen R, Mortimer D, editors. Toward reproductive certainty: fertility and genetics beyond. London: Parthenon Publishing; 1999. p. 378–88.Google Scholar
- 34.Kai Y, Mio Y. Multidisciplinary analyses of human 1PN zygotes using a high-resolution time-lapse cinematography system and molecular biological techniques. J Mamm Ova Res. 2016;1:25–9.Google Scholar