Human haploid parthenotes (embryos built from an artificially activated unfertilized oocyte, maternal genome only) develop with a markedly prolonged second cell cycle compared with biparental embryos, while haploid androgenotes (paternal genome only in an enucleated oocyte) show unusually short cell cycles. Since the 2016 morphokinetic study that established these differences, later work has resolved several of its open questions: uniparental embryos can reach the blastocyst stage (parthenotes directly; androgenetic embryos in diploid form after pronuclear transfer), embryonic genome activation in androgenotes has been characterized at single-cell resolution, and transcriptomes plus blastocyst-stage methylomes of uniparental embryos are now available. The surviving open core is mechanistic: the cause of the prolonged second cell cycle in parthenotes — absence of the sperm-derived centrosome, parent-of-origin DNA methylation dynamics, altered transcriptional activation, or a combination — has still not been discriminated, because no study has combined subphase-resolved first/second cell-cycle kinetics with direct methylation and transcriptome profiling of the same haploid uniparental embryos at the 1-to-4-cell stage, nor performed causal perturbation. Whether strictly haploid androgenotes can progress beyond day 3 to the blastocyst also remains untested.

Background

Early human development can be modeled with uniparental constructs. A parthenogenote (or gynogenetic embryo) is produced by artificially activating an unfertilized metaphase-II oocyte — the mature egg, arrested partway through meiosis — for example with a calcium ionophore plus puromycin, so that development proceeds with a maternal genome only; an androgenote is produced by injecting a single sperm into an enucleated oocyte (intracytoplasmic sperm injection, ICSI), so it contains a paternal genome only. Because only one genome copy is present, these constructs are haploid, and they are never used for reproduction; they are research models for dissecting what each parental genome contributes to the earliest stages. Two parental asymmetries are central. First, in humans (unlike rodents) the centrosome — the microtubule-organizing center that assembles the mitotic spindle — is contributed by the sperm at fertilization, so a parthenote develops without a sperm centrosome; a single-sperm androgenote carries the normal single sperm centriole, whereas a diploid androgenote made by injecting two sperm into one oocyte carries an extra centriole, which causes multipolar spindles and abnormal division. Second, mammalian genomes carry genomic imprinting, parent-of-origin-specific DNA methylation marks, so maternal-only and paternal-only embryos begin with different epigenetic landscapes. Development is initially driven by maternally stored messenger RNA; the takeover by transcription from the embryo's own genome is called embryonic genome activation (EGA), which occurs as a minor wave in the first cell cycles and a major wave around the 4-to-8-cell stage in humans. In time-lapse morphokinetics (continuous microscopy with annotated cleavage timestamps), the duration of the second cell cycle, cc2, is measured as the interval between the second and third cleavage divisions, and cc3 analogously for the third cell cycle. Escribá et al. (2016) compared the first seven cleavages of 8 haploid parthenotes, 10 haploid androgenotes, and 20 euploid (chromosomally normal) biparental embryos. Every cleavage occurred later in parthenotes; cc2 was significantly longer in parthenotes than in both androgenotes and biparental embryos, while androgenotes had a consistently shorter cc3. Culture was stopped at the 6-to-10-cell stage (day 3), and the study's kinetics-only design left several questions unresolved: (i) whether haploid parthenotes or androgenotes can progress to the blastocyst stage — the hollow day-5–6 embryo containing the inner cell mass (the embryo-proper precursor) and the trophectoderm (the precursor of the placenta); (ii) what causes the longer cc2 in parthenotes, since first-cell-cycle subphases, methylation profiles, and transcriptome profiles were not analyzed — the data could not distinguish among the absence of the sperm centrosome, different parental DNA methylation dynamics, altered transcriptional activation, or a combination; (iii) whether androgenotes undergo major EGA in the same developmental window as biparental embryos and how they sustain early development under short, highly synchronous cell cycles; and (iv) the transcriptomic and epigenetic programs of these embryos at the earliest stages. Between 2016 and 2026, items (i) and (iii) were largely settled and much of (iv) was mapped: haploid human embryonic stem cell lines were derived from parthenogenetically activated haploid oocytes (showing parthenotes can form blastocysts); diploid androgenetic embryos produced by male pronuclear transfer — a design that avoids the extra centriole — were shown to reach blastocysts with identifiable inner cell mass and trophectoderm, with genome activation and blastocyst methylomes analyzed; and single-cell RNA sequencing series profiled haploid androgenotes and parthenogenotes through the first four cell cycles, establishing that both show reduced genome activation over the first three cycles, that parthenogenotes spike at the fourth cycle, and that key transcription factors (LEUTX, DUXA, DUX4, ZNF675) drive the paternal contribution. What none of these studies provided is subphase-resolved kinetics of the first and second cell cycles, direct methylation profiles of haploid uniparental embryos at the 1-to-4-cell stage (existing methylomes cover diploid uniparental blastocysts), or a causal test such as reconstituting the sperm centrosome in a parthenote.

Problem Statement

Determine the mechanism responsible for the prolonged second cell cycle (cc2) of human haploid parthenogenetic embryos relative to biparental embryos and androgenotes, as observed by Escribá et al. (2016). Specifically, discriminate among — or establish a combination of — (i) the absence of the sperm-derived centrosome and the associated spindle/microtubule deficits, (ii) parent-of-origin-specific DNA methylation dynamics of the oocyte-only epigenome, and (iii) altered timing or magnitude of transcriptional activation (minor/major embryonic genome activation). Also determine whether strictly haploid androgenotes can progress beyond day 3 to form a blastocyst, since blastocyst development has so far been demonstrated only for parthenogenetic/gynogenetic embryos and for androgenetic embryos in diploid (pronuclear-transfer) form, and culture of strictly haploid androgenetic bioconstructs was legislatively limited to day 3. Characterizing the epigenetic and transcriptional programs of these cleavage-stage uniparental embryos is part of this mechanistic discrimination.

References

  1. Escribá MJ, Escrich L, Galiana Y, Grau N, Galán A, Pellicer A. Kinetics of the early development of uniparental human haploid embryos. Fertility and Sterility 2016;105(5):1360–1368.e1. doi:10.1016/j.fertnstert.2015.12.139, https://doi.org/10.1016/j.fertnstert.2015.12.139
  2. Sagi I, Chia G, Golan-Lev T, Peretz M, Weissbein U, Sui L, Sauer MV, Yanuka O, Egli D, Benvenisty N. Derivation and differentiation of haploid human embryonic stem cells. Nature 2016;532(7597):107–111. doi:10.1038/nature17408, https://doi.org/10.1038/nature17408
  3. Leng L, Sun J, Huang J, Gong F, Yang L, Zhang S, Yuan X, Fang F, Xu X, Luo Y, Bolund L, Peters BA, Lu G, Jiang T, Xu F, Lin G. Single-Cell Transcriptome Analysis of Uniparental Embryos Reveals Parent-of-Origin Effects on Human Preimplantation Development. Cell Stem Cell 2019;25(5):697–712.e6. doi:10.1016/j.stem.2019.09.004, https://doi.org/10.1016/j.stem.2019.09.004
  4. Xu J, Shu Y, Yao G, Zhang Y, Niu W, Zhang Y, Ma X, Jin H, Zhang F, Shi S, Wang Y, Song W, Dai S, Cheng L, Zhang X, Xie W, Hsueh AJW, Sun Y. Parental methylome reprogramming in human uniparental blastocysts reveals germline memory transition. Genome Research 2021;31(9):1519–1530. doi:10.1101/gr.273318.120, https://doi.org/10.1101/gr.273318.120
  5. Li C, Zhang Y, Leng L, Pan X, Zhao D, Li X, Huang J, Bolund L, Lin G, Luo Y, Xu F. The single-cell expression profile of transposable elements and transcription factors in human early biparental and uniparental embryonic development. Frontiers in Cell and Developmental Biology 2022;10:1020490. doi:10.3389/fcell.2022.1020490, https://doi.org/10.3389/fcell.2022.1020490
  6. de Castro P, Vendrell X, Escrich L, Grau N, Gonzalez-Martin R, Quiñonero A, Dominguez F, Escribá MJ. Comparative single-cell transcriptomic profiles of human androgenotes and parthenogenotes during early development. Fertility and Sterility 2023;119(4):675–687. doi:10.1016/j.fertnstert.2022.12.027, https://doi.org/10.1016/j.fertnstert.2022.12.027
  7. Vendrell X, de Castro P, Escrich L, Grau N, Gonzalez-Martin R, Quiñonero A, Escribá MJ, Domínguez F. Longitudinal profiling of human androgenotes through single-cell analysis unveils paternal gene expression dynamics in early embryo development. Human Reproduction 2024;39(6):1186–1196. doi:10.1093/humrep/deae072, https://doi.org/10.1093/humrep/deae072 (open access full text at PMC11145015)
  8. Vendrell X, De Castro P, Escrich L, Grau N, González-Martín R, Quiñonero A, Galán A, Domínguez F, Escribá MJ. Transcriptomic profiles of human parthenogenotes: contribution of the maternal genome across the early embryo development. F&S Science 2026;7(2):150–162. doi:10.1016/j.xfss.2025.12.003, https://doi.org/10.1016/j.xfss.2025.12.003