Comparison of two methodologies of oocyte enucleation

Grau, N.; Escrich, L.; Albert, Carmela; Delgado, Arantza; Santos, María José de los; Escribá, Marı́a José

doi:10.1016/j.fertnstert.2011.07.948

Cited by 3 publications

(2 citation statements)

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“…For MII oocyte enucleation, 30 surviving, warmed oocytes were inspected for the presence of the meiotic spindle using a light microscope equipped with Octax PolarAid (Olympus) imaging software. Once detected, the spindle was removed by gentle aspiration through an ICSI pipette (Cook) according to the procedure described by Grau et al (44). One hour later, absence of the spindle was confirmed in the 21 oocytes that had survived manipulation (70.0% successful enucleation rate).…”

Section: Haploid Androgenote Productionmentioning

confidence: 99%

Kinetics of the early development of uniparental human haploid embryos

Escribá

Escrich

Galiana

et al. 2016

Fertility and Sterility

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Section: Haploid Androgenote Productionmentioning

confidence: 99%

Kinetics of the early development of uniparental human haploid embryos

Escribá

Escrich

Galiana

et al. 2016

Fertility and Sterility

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“…AGs were created using established protocols ( Kono et al , 1993 , 1995 ) with slight modifications ( Escribá et al , 2016 ). Briefly, MII oocytes were enucleated by polarized light microscopy (Oosight ® ; Barcelona, Spain) ( Grau et al , 2011 ), followed by fertilization by ICSI with donor sperm. The resulting AG embryos, with a single pronucleus and without a second polar body in the perivitelline space (n = 14; 82% fertilization rate), were cultured for an additional three days, as a maximum.…”

Section: Methodsmentioning

confidence: 99%

Longitudinal profiling of human androgenotes through single-cell analysis unveils paternal gene expression dynamics in early embryo development

Vendrell,

de Castro,

Escrich

et al. 2024

Human Reproduction

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STUDY QUESTION How do transcriptomics vary in haploid human androgenote embryos at single cell level in the first four cell cycles of embryo development? SUMMARY ANSWER Gene expression peaks at the fourth cell cycle, however some androcytes exhibit unique transcriptional behaviors. WHAT IS KNOWN ALREADY The developmental potential of an embryo is determined by the competence of the oocyte and the sperm. However, studies of the contribution of the paternal genome using pure haploid androgenotes are very scarce. STUDY DESIGN, SIZE, DURATION This study was performed analyzing the single-cell transcriptomic sequencing of 38 androcytes obtained from 10 androgenote bioconstructs previously produced in vitro (de Castro et al., 2023). These results were analyzed through different bioinformatics software such as g: Profiler, GSEA, Cytoscape, and Reactome. PARTICIPANTS/MATERIALS, SETTING, METHODS Single cell sequencing was used to obtain the transcriptomic profiles of the different androcytes. The results obtained were compared between the different cycles studied using the DESeq2 program and functional enrichment pathways using g: Profiler, Cytoscape, and Reactome. MAIN RESULTS AND THE ROLE OF CHANCE A wave of paternally driven transcriptomic activation was found during the third-cell cycle, with 1128 upregulated and 225 downregulated genes and the fourth-cell cycle, with 1373 upregulated and 286 downregulated genes, compared to first-cell cycle androcytes. Differentially expressed routes related to cell differentiation, DNA-binding transcription, RNA biosynthesis and RNA polymerase II transcription regulatory complex, and cell death were found in the third and fourth with respect to the first-cell cycle. Conversely, in the fourth cell cycle, 153 downregulated and 332 upregulated genes were found compared with third cell cycle, associated with differentially expressed processes related to E-box binding and zinc finger protein 652 (ZNF652) transcription factor. Further, significant overexpression of LEUTX, PRAMEF1, DUXA, RFPL4A, TRIM43, and ZNF675 found in androgenotes, compared to biparental embryos, highlights the paternal contributions to zygote genome activation. LARGE SCALE DATA All raw sequencing data are available through the Gene Expression Omnibus (GEO) under accessions number: GSE216501. LIMITATIONS, REASONS FOR CAUTION Extrapolation of biological events from uniparental constructs to biparental embryos should be done with caution. Maternal and paternal genomes do not act independently of each other in a natural condition. The absence of one genome may affect gene transcription of the other. In this sense, the haploid condition of the bioconstructs could mask the transcriptomic patterns of the single cells. WIDER IMPLICATIONS OF THE FINDINGS The results obtained demonstrated the level of involvement of the human paternal haploid genome in the early stages of embryo development as well as its evolution at the transcriptomic level, laying the groundwork for the use of these bioconstructs as reliable models to dispel doubts about the genetic role played by the paternal genome in the early cycles of embryo development. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by Instituto de Salud Carlos III (ISCIII) through the project ‘PI22/00924’, co-funded by European Regional Development Fund (ERDF); ‘A way to make Europe’. F.D. was supported by the Spanish Ministry of Economy and Competitiveness through the Miguel Servet program (CPII018/00002). M.J.E. was supported by Instituto de Salud Carlos III (PI19/00577 [M.J.E.]) and FI20/00086. P.dC. was supported by a predoctoral grant for training in research into health (PFIS PI19/00577) from the Instituto de Salud Carlos III. All authors declare having no conflict of interest with regard to this trial.

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