Developing In Vitro Models of Human Embryonic Aneuploidy
Authors:   , Stepney Ellie 
Published: February 2023

<p><b>Approximately half of the human embryos produced by in vitro fertilisation (IVF) worldwidecontain an incorrect number of chromosomes (aneuploidy). Preimplantation genetic testing foraneuploidy (PGT-A) is available but relies on a biopsy taken from an embryo's trophectodermcell layer, which is invasive, expensive, and time-consuming. The development of a non-invasivePGT-A that utilises the genetic material secreted from an embryo to determine its ploidy status ishighly desirable. The development of such a test relies on good laboratory models of aneuploidy.</b></p>
<p>Triploid embryos, which contain an entire extra chromosome set, are a common form ofaneuploidy and several models can be developed in the laboratory. One such model is digynictriploid embryos that have an extra maternal haploid chromosome set by manipulating meiosis.</p>
<p>Herein, the in vitro generation of digynic triploid embryos was developed and optimised byexposing mouse oocytes to 10 μg/mL of cytochalasin-B for four hours, beginning three hoursafter conventional IVF. This blocks expulsion of the second polar body resulting in an embryowith one paternal and two maternal sets of chromosomes. These embryos developed through tothe blastocyst stage.</p>
<p>A second model is diandric triploid embryos that have one maternal and two paternal sets ofchromosomes through dispermy. In this study, two spermatids were injected into a single maturemouse oocyte using intracytoplasmic sperm injection (ICSI). Diandric triploidy was lesscompatible with embryonic development, as observed by the inability of these embryos to cleavebeyond the two-cell stage.</p>
<p>A model of parthenogenetic activation was also characterised after several zygotes presentedwith one pronucleus following conventional IVF, in the presence and absence of cytochalasin-Bincubation, or insemination by ICSI, and proved capable of developing to the blastocyst stage.</p>
<p>The developmental progression of single pronucleated, chromosomally normal (euploid), anddigynic triploid embryos was recorded using Primo VisionTM cameras enabling early embryoniccytokinetic events to be compared. We found no morphokinetic parameters which may act aspredictors of aneuploidy. Though, the timeliness in which an embryo initiates and completescleavage into a three-cell and eight-cell embryo, as well as the time taken to cleave into a four-6cell or morula embryo, may be predictors of the developmental potential of mouse embryos,regardless of ploidy status.</p>
<p>In addition, gene expression analyses of single pronucleated, euploid, and digynic triploidmorula and blastocyst embryos by qPCR revealed no significant differences in the mRNAexpression of Phlda2, Grb10, or Pdcd4. However, the expression of Igf2 mRNA wassignificantly higher in single pronucleated blastocysts compared to euploid and digynic triploidembryos.</p>
<p>Overall, this study characterised in vitro laboratory models of human embryonic digynictriploidy and parthenogenetic activation in mice and was successful in producing diandrictriploid mouse embryos for use in future aneuploidy studies.</p>