The 2016 demonstration that the entire mouse female germ-line cycle can be completed in a dish — from pluripotent stem cells through fertilization-competent MII oocytes to live offspring, and back to pluripotent stem cell lines — established in vitro gametogenesis (IVG) as a real technology, but the oocytes it produces remain far inferior to in vivo oocytes: full-term development after in vitro fertilization (IVF) occurs at well under one percent (0.9%, 26/2,753 transferred 2-cell embryos, reported) versus the majority for in vivo oocytes (61.7%, 37/60), roughly half of pachytene cells in vitro show meiotic chromosome asynapsis (53.8% reported) and 22.2% of the resulting MII oocytes are aneuploid, and single-oocyte profiling reveals transcriptome misregulation (including Polycomb-target genes and long-terminal-repeat (LTR) retrotransposon classes) and epigenetic maturation defects (abnormal 5-hydroxymethylcytosine (5hmC) acquisition, failure at zygotic genome activation). One element of the original 2016 challenge — replacing embryonic gonadal somatic cells with pluripotent-stem-cell-derived somatic support — was substantially resolved in 2021 by FOSLC-based reconstituted follicles that yielded live offspring, and an ovarian-soma-free culture now reaches germinal-vesicle-stage oocyte-like cells. The surviving open problem is the quality gap itself: identify the causes of the documented meiotic, transcriptomic and epigenetic defects, correct them, and obtain fertilization-competent MII oocytes that yield healthy fertile offspring at rates approaching matched in vivo controls — in any in vitro system for PSC-derived mouse oogenesis and by any defined intervention — with validated quality metrics for IVG oocytes as a related, source-recognized desideratum.

Background

In vitro gametogenesis (IVG) is the attempt to generate functional gametes from pluripotent stem cells in culture. In the mouse female line, the pivotal protocol (Hikabe et al., Nature 539:299–303, 2016) worked as follows. Embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs, pluripotent cells reprogrammed from fibroblasts) were first differentiated into primordial germ cell-like cells (PGCLCs), the culture equivalent of nascent primordial germ cells. PGCLCs were then aggregated with embryonic (E12.5, embryonic day 12.5) ovarian somatic cells dissected from fetuses; the aggregate self-organizes into a 'reconstituted ovary' (rOvary) in which the PGCLCs enter meiotic prophase I, form primary follicles, and — after a multi-step follicle culture and in vitro maturation — yield metaphase II (MII) oocytes, the fertilization-arrested egg stage. These IVG oocytes could be fertilized by in vitro fertilization (IVF) and a small fraction supported full-term development after embryo transfer; new pluripotent stem cell lines re-derived from those IVG embryos (rESCs) completed the entire female germ-line cycle in vitro.

The 2016 report itself quantified how poor the in vitro oocytes are relative to in vivo ovulated oocytes: only 0.9% (26/2,753) of transferred 2-cell embryos derived from IVG oocytes developed to term versus 61.7% (37/60) for oocytes from superovulated mice, and oocytes within rOvaries showed defective meiotic chromosome pairing — asynapsis (failure of homologous chromosomes to fully synapse along the synaptonemal complex, the protein structure that links homologs during meiotic prophase) at the pachytene stage in 53.8% of cells in vitro, with aneuploidy (gametes with abnormal chromosome number) in 22.2% of the resulting MII oocytes. Transcriptome analysis found differentially expressed genes enriched for mitochondrial function in MII oocytes and abnormal dynamics of LTR retrotransposons (endogenous retroviral elements regulated during germ-cell development) at the secondary follicle stage. The paper's discussion posed five outstanding problems: (i) the mechanistic causes of low developmental competence; (ii) how the molecular perturbations arise and how culture conditions (e.g., metabolic supplementation, oxygenation) should be changed; (iii) how to improve meiotic synapsis and reduce aneuploidy; (iv) replacement of the exogenous embryonic gonadal somatic cells with bona fide pluripotent-stem-cell-derived somatic cells, enabling a PSC-only workflow; and (v) systematic quality metrics for heterogeneous IVG oocytes and derived stem cell lines.

Subsequent work transformed parts of this agenda. A defined-culture analysis identified eight transcription factors sufficient to trigger oocyte growth, and their enforced expression converted pluripotent stem cells directly into fertilization-competent oocyte-like cells without primordial germ cell specification or meiosis (Hamazaki et al., Nature 589:264–269, 2020) — a demonstration of regulative potential, though not of meiotically normal eggs. Yoshino et al. (Science 373:eabe0237, 2021) recreated the fetal ovarian somatic lineage in vitro: ESC-derived fetal ovarian somatic cell-like cells (FOSLCs), aggregated with PGCLCs, formed reconstituted follicles in which PGCLCs entered meiosis and generated oocytes capable of fertilization and development to live offspring — substantially resolving item (iv), albeit at low efficiency and with oocytes of reduced quality. On the mechanism of poor quality, Aizawa et al. (EMBO Journal 42:e113955, 2023) compared each culture step against in vivo development and showed that genes normally upregulated during oocyte growth, many of them Polycomb targets (genes repressed by the Polycomb chromatin machinery), are preferentially misregulated during in vitro oogenesis; that structurally normal IVG oocytes nevertheless fail at zygotic genome activation (ZGA, the onset of embryonic transcription after fertilization); and that they acquire 5-hydroxymethylcytosine (5hmC, an oxidized form of methylated cytosine produced by TET enzymes) abnormally on maternal chromosomes. Most recently, Nosaka et al. (Developmental Cell 60:2976–2994, 2025) established an ovarian-soma-free (OSC-free) system in which retinoic acid and BMP signaling on feeders drive PGCLCs through meiotic prophase I to diplotene arrest, and defined cytokines, signaling activators, and antioxidants then support growth to germinal-vesicle (GV, prophase-I-arrested fully grown oocyte) -stage oocyte-like cells able to resume meiosis — but without reaching MII, fertilization, or offspring. Reviews through 2024–2026 (Aizawa et al., BioEssays 2025; Yang et al., Clinical Epigenetics 18:25, 2026) concur that meiotic and epigenetic abnormalities of IVG gametes persist and that oocyte quality is the central remaining limitation, while on the human side PGCLC-derived cells arrest before meiosis (Mizuta et al., EMBO Journal 2022; Murase et al., Nature 631:170–178, 2024; Smela et al., Science Advances 11:eadu0384, 2025), keeping clinical translation distant (Ledford, Nature 646:534–536, 2025; Landecker & Clark, Nature Biotechnology 44:518–520, 2026).

Problem Statement

Starting from the 2016 demonstration of full in vitro oogenesis in the mouse and the subsequent resolution of the PSC-derived somatic-niche sub-problem, the open problem is to close the quality gap that separates in vitro-generated (IVG) oocytes from in vivo oocytes: make oocytes derived from pluripotent stem cells in culture developmentally competent at rates approaching in vivo oocytes. Concretely, the objective is to establish the causes of the documented defects — meiotic chromosome asynapsis and consequent aneuploidy during in vitro meiotic prophase, misregulation of growth-phase genes including Polycomb targets and LTR retrotransposon classes, and abnormal epigenetic maturation including 5hmC acquisition and zygotic genome activation failure — that limit developmental competence to term, to correct them, and to show that the corrected system yields fertilization-competent MII oocytes whose full-term development rate, meiotic synapsis fidelity, transcriptome, and epigenome approach matched in vivo controls and whose offspring are healthy and fertile. Neither the intervention class nor the culture system is restricted: corrections may be defined modifications of the culture system, modifications of the somatic niche, or genetic, epigenetic or cytoplasmic interventions of the kinds the field is exploring; and the work may be carried out in any in vitro system for PSC-derived mouse oogenesis — reconstituted ovaries made with embryonic ovarian somatic cells (the system in which these defects were quantified), fully PSC-derived reconstituted follicles, or an ovarian-soma-free culture, which currently reaches only germinal-vesicle stage. The objective also includes demonstrating causality by intervention rather than correlation, and deriving validated quality metrics that prospectively distinguish competent from incompetent IVG oocytes and explain any residual gap. The target is for the full-term development rate, meiotic synapsis fidelity, transcriptome, and epigenome of the corrected oocytes to approach matched in vivo controls.

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