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

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.

Do immature and mature sibling oocytes recovered from stimulated cycles have the same reproductive potential?

RESEARCH QUESTION: How can laboratory and clinical outcomes of spontaneously, early maturing germinal-vesicle oocytes and sibling in-vivo-matured (metaphase II [MII]) oocytes be quantified and compared? DESIGN: A prospective, non-randomized intra-cohort study of oocytes from women aged 38 years or younger, with six or fewer MII oocytes and four or more germinal vesicles retrieved. No indication was identified for genetic tests or oocyte or embryo cryopreservation. The study was carried out at IVIRMA-Valencia. Early maturing germinal vesicles were selected for reproductive purposes. In vitro- and in-vivo MII oocytes were fertilized. After time-lapse culture, hatching blastocysts from germinal vesicles were biopsied for aneuploidy screening and vitrified. Laboratory and clinical outcomes were compared according to oocyte origin. RESULTS: Almost 70% of germinal vesicles had matured early and spontaneously, and had comparable in vitro-outcomes and morphokinetics to sibling in vivo-matured oocytes. Fifty per cent of biopsied blastocysts were euploid. Germinal-vesicle rescue increased the number of MII oocytes per cycle to 3.9, finally adding one extra-blastocyst per cycle. A live birth confirmed the feasibility of this approach. Further data, however, are needed to quantify its real contribution to standard intracytoplasmic sperm injection cycles. Nevertheless, 40% of patients obtained either an immediate advantage (reduction of cancellation rate) or long-term benefit (availability of extra blastocysts of attempts). CONCLUSIONS: Germinal-vesicle rescue can be considered as a complementary approach when folliculometry (expected) and number of MII (observed) are unequal.