P-803 Reconstructing oocytes via haploidization: Challenges in development, ploidy, and chromosome dynamics
W Vandenberghe, A Rybouchkin, M Loosveldt, A Mørch, D Stoop, S Chuva de Sousa Lopes, B Menten, B HeindryckxAbstract
Study question
Is the efficiency of creating euploid embryos by haploidization affected by genetic background and source of donor cell?
Summary answer
Euploid embryos can be obtained irrespective of genetic background, but aberrant spindle formation affects developmental competence in all groups.
What is known already
Women without functional oocytes currently have to rely on oocyte donation or adoption. Haploidization represents an alternative approach, by reducing a diploid genome by half, allowing the conception of a child that is genetically related to both parents. Live mouse offspring has previously been obtained following haploidization of cumulus cells, but both pre- and post-implantation efficiency remain poor. Moreover, contradictions regarding the genetic background of the donor cell and its ability to be haploidized have arisen. To date, no comprehensive live imaging of chromosome dynamics has been conducted, leaving uncertainties regarding the quality of spindle formation and chromosome behavior unresolved.
Study design, size, duration
The developmental competence and euploidy of mouse pre-implantation embryos following haploidization was assessed by comparing different donor cells. Genetic background effects were examined by comparing B6D2F1 and C57BL/6 donor cells, while the impact of the donor cell type was investigated by haploidizing cumulus cells versus embryonic stem cells (ESC). To explore spindle and chromosome dynamics, reconstructed oocytes underwent confocal live imaging.
Participants/materials, setting, methods
A total of 314 mouse MII oocytes were enucleated and injected with B6D2F1 cumulus cells (n = 131), B6 cumulus cells (n = 115), or G1 synchronized ESC (n = 68). After two hours, reconstructed oocytes were fertilized via piezo-ICSI, extruding half of the somatic chromosomes. Controls included 119 non-enucleated oocytes fertilized by piezo-ICSI and 88 oocytes activated by SrCl2. Embryo development was tracked to the blastocyst stage (96h). Spindle-chromosome complexes (n = 49) were live-imaged, and 31 embryos underwent CNVseq ploidy analysis.
Main results and the role of chance
Embryos resulting from haploidization showed 76% 2-cell formation (66% for B6D2F1 cumulus cells; 97% for B6 cumulus cells; 62% for B6 ESCs), similar to 88% for sperm-injected MII oocytes and 80% for SrCl2 activated oocytes. Only two reconstructed oocytes, (one injected with B6D2F1 cumulus cell and one injected with B6 ESC) , developed into blastocysts. In contrast, sperm-injected MII oocytes showed a blastocyst rate of 58%, while parthenogenetic embryos showed a blastocyst rate of 94%. CNVseq of embryos derived from haploidization revealed severe aneuploidies, indicating random segregation of somatic chromosomes after fertilization in the majority of analyzed samples. Intriguingly, euploidy was detected in 11% of embryos with B6D2F1 donor cells and 14% of embryos with B6 donor cells (16% for cumulus cells; 0% for ESCs). Live imaging of reconstructed oocytes revealed rapid condensation of chromosomes (<30min) but chaotic spindle formation two hours after reconstruction. Only those injected with B6 cells formed bipolar spindles with nearly aligned chromosomes on the metaphase plate. Extended live imaging showed that alignment of all chromosomes at the metaphase plate was complete at the earliest at 4 hours after reconstruction, before sperm injection.
Limitations, reasons for caution
Currently, the study has not yet incorporated transcriptome or proteome analysis. Therefore, it remains to be determined whether impaired development results from insufficient reprogramming or is solely due to aneuploidy.
Wider implications of the findings
Our results show that haploidization can produce euploid embryos, but pre-implantation development remains poor, even when using ESCs, which have a more reprogrammed epigenetic landscape. We demonstrate that haploidization is not limited to inbred mice, as euploidy is achieved with a hybrid genetic background, supporting potential translation to humans.
Trial registration number
No