The construct, which the team calls SPHEGaT — spheroids of endocrine granulosa and theca cells — was built from primary human cells with careful attention to spatial organization. Granulosa cells, harvested from patients undergoing in vitro fertilization, expressed the aromatase enzyme CYP19A1 and the follicle-stimulating hormone receptor, marking them as the estrogen-producing core. Theca-like cells were differentiated from ovarian stromal cells isolated from postmenopausal donors, and they expressed CYP17A1 and CD13, consistent with their role in producing the androgens that granulosa cells then convert into estradiol. When the granulosa cells were seeded into low-attachment plates, they spontaneously self-aggregated into compact spheres within about three days. Theca-like cells were then added and progressively organized around the granulosa core, and a thin layer of collagen type I was deposited between the two compartments to mimic the follicular basement membrane. The result was a multilayered structure that echoes the compartmental arrangement of a native follicle.

Architecture, it turns out, is not merely cosmetic. After nine days of culture, the spheroids were remarkably healthy: quantitative analysis showed that 97.45 percent of the spheroid area remained viable, with no detectable focal necrosis. Dense three-dimensional aggregates often suffer from oxygen starvation in their cores, a problem the team checked directly by staining for HIF-2α, a marker of hypoxic stress. Only about 3 percent of nuclei were positive, and three-dimensional reconstruction confirmed the absence of a hypoxic core. Critically, the spheroids were functionally competent: the culture medium contained endogenous levels of estradiol at roughly 65 picograms per milliliter and progesterone at roughly 186 nanograms per milliliter. The spatially organized assembly had produced a living, breathing endocrine unit — one that secretes hormones the way a follicle does, rather than sitting passively in a bath of supplied steroids.

The decisive experiment came when the researchers coupled these spheroids to endometrial organoids, miniature gland-like structures grown from endometrial biopsies of women in their late twenties. Over six days of co-culture, the organoids underwent a visible transformation. Monocultured organoids retained mostly spherical, cyst-like shapes, but organoids exposed to the ovarian spheroids progressively developed folded, lobulated, and optically denser architectures. The proportion of folded organoids rose from about 2.4 percent in monoculture to nearly 13.7 percent in co-culture by day six, a highly significant difference. Circularity and solidity — geometric measures of how round and how smooth an organoid is — both declined over time in the co-cultured condition, indicating the emergence of irregular, invaginated epithelial structures reminiscent of glandular remodeling in the cycling endometrium.

What makes this remodeling striking is that it was not driven by proliferation. Ki67 staining, a standard marker of dividing cells, showed no significant difference between monocultured and co-cultured organoids, and total organoid area grew comparably in both conditions. The ovarian spheroids were not making the tissue grow faster; they were teaching it to reorganize. This mirrors in vivo physiology, where epithelial proliferation peaks during the estrogen-dominated proliferative phase and then stabilizes as progesterone steers the tissue toward secretory maturation. A trend toward reduced vimentin expression in co-cultured organoids further hinted at a dampening of epithelial-mesenchymal plasticity, a shift associated with secretory differentiation. In short, the ovarian signal was instructing architecture, not cell division.

At the molecular level, the co-culture produced clear evidence of endocrine reprogramming. Progesterone receptor expression surged in co-cultured organoids: only about 4.7 percent of nuclei were progesterone receptor-positive in monoculture, compared with roughly 40.5 percent after exposure to the spheroids. Estrogen receptor levels, by contrast, remained unchanged. The co-cultured organoids also upregulated a selective set of progesterone-responsive, secretory-phase genes, including PAEP, which encodes pregnancy-associated endometrial protein; SPP1, or secreted phosphoprotein 1; and HSD17B2, an enzyme involved in steroid metabolism. PAEP secretion, measured by ELISA, rose progressively in co-culture. Other receptivity-associated genes such as LIF, SOX17, GATA2, and AREG did not change significantly, indicating that the organoids entered a partial, progesterone-driven secretory state rather than acquiring the full implantation-window program — a limitation the authors attribute in part to the absence of an estrogen-priming phase in the current protocol.

One of the most intriguing observations was a hint of bidirectional communication. In the co-culture system, progesterone levels in the medium dropped markedly — to about 68 nanograms per milliliter compared with roughly 239 in spheroid monocultures — while estradiol levels stayed stable. This hormone-specific reduction suggests the endometrium may be actively metabolizing, taking up, or otherwise modulating ovarian progesterone output, echoing the known ability of uterine prostaglandins and other mediators to regulate luteal steroid production in vivo. The platform, in other words, does not just deliver hormones to the endometrium; it establishes a coupled endocrine axis in which hormone availability reflects ongoing integration between the two tissue compartments.

To test whether the morphogenic effects depended on steroids alone, the team performed an elegant depletion experiment. They treated spheroid-conditioned medium with dextran-coated charcoal, stripping out roughly 59 percent of the estradiol and 90 percent of the progesterone, and then compared three conditions: intact conditioned medium, depleted medium, and depleted medium with exogenous hormones added back. The results were unambiguous. Untreated conditioned medium induced the greatest epithelial folding over time. Depleted medium still supported a reduced but significant increase in folding — hinting at residual steroid activity or non-steroidal factors — but static hormone add-back failed entirely to restore the morphogenic phenotype and actually produced the lowest folding response of all. Organoids in the add-back condition grew larger but stayed smoother, suggesting that supraphysiologic or imbalanced steroid exposure biases tissue toward expansion rather than differentiation.

The implication is profound: the ovary’s instructive power over the endometrium cannot be reduced to a prescription of estradiol and progesterone. The intact follicle is a complex endocrine and paracrine factory, co-secreting inhibins and activins, growth factors such as EGF-like ligands and basic FGF, cytokines, metabolites, carrier proteins, and extracellular vesicles loaded with proteins, lipids, and regulatory RNAs. Many of these factors have been implicated in endometrial remodeling and implantation, and the temporal fluctuations, concentration gradients, and local metabolism inherent to an intact ovarian unit may be as important as any single molecule. Static hormone replacement, the study suggests, fundamentally cannot reproduce that context.

The researchers are candid about the limitations. The system models a progesterone-dominant environment without a preceding estrogen-priming phase, so it captures only part of the menstrual cycle’s proliferative-to-secretory transition. The co-culture lacks stromal decidualization, immune cells, vascular elements, and embryo-derived signals, all of which contribute to true uterine receptivity. The theca-like cells derive from postmenopausal donors, raising questions about age-related epigenetic carryover, and charcoal-dextran treatment may have altered non-steroidal components of the conditioned medium. Yet these caveats do not diminish the central achievement. The team has built the first spatially organized, fully human ovarian endocrine construct capable of directing endometrial morphogenesis in vitro, and shown that its effects exceed anything achievable with hormones alone. The platform opens a tractable window onto infertility, implantation failure, progesterone resistance, and endocrine disruption — and points toward a future generation of reproductive models built not on hormone recipes, but on reconstructed, talking tissues.

Subject of Research: Engineering spatially organized human ovarian spheroids to model ovarian-endometrial endocrine communication in vitro

Article Title: Spatially Organized Human Ovarian Spheroids Instruct Endometrial Morphogenesis

Article References: Sousa, M. J., Vriendt, S. D., Liu, L., Ruiz, T. F. R., Vankelecom, H., & Amorim, C. A. (2026). Spatially Organized Human Ovarian Spheroids Instruct Endometrial Morphogenesis. Advanced Science, 13(55), Article e76538. https://doi.org/10.1002/advs.76538

Image Credits: AI Generated

DOI: 10.1002/advs.76538

Keywords: ovarian spheroids, endometrial organoids, granulosa cells, theca-like cells, progesterone signaling, estradiol, steroidogenesis, reproductive biology, organ-on-a-chip, endometrial morphogenesis, infertility modeling, 3D cell culture