Integrative single-cell transcriptomics and experimental validation reveal that Tusizi Formula ameliorates premature ovarian insufficiency by preserving the primordial follicle pool and promoting granulosa cell differentiation.

BACKGROUND: Premature ovarian insufficiency (POI) is characterized by reduced ovarian reserve and disrupted folliculogenesis, often driven by granulosa cell (GC) dysfunction. Traditional Chinese medicine formula TSZF has been widely used for POI, yet its mechanisms and active components remain unclear. This study aimed to elucidate the mechanisms by which TSZF remodels the ovarian microenvironment to counteract POI and to explore its active components.

METHODS: The composition of the TSZF was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Tipterygium glycosides (TG) induced POI rat model was established. We evaluated TSZF's ovarian-protective effects via estrous cyclicity, serum hormones and ovarian histopathology. scRNA-seq and qRT-PCR explored and validated its cellular mechanisms. Primordial follicle isolation and culture supported its protective role in follicle reserve maintenance. In vitro experiments and molecular docking validated the ovarian‑protective effects of its active components.

RESULTS: TSZF restored estrous cyclicity, increased primordial/primary/antral follicles, reduced atretic follicles, and normalized E2, AMH, FSH, and LH levels. scRNA-seq revealed that TSZF was associated with enhanced progenitor GC differentiation into cumulus and mural GC subtypes, a key step for antral follicle development. This was accompanied by upregulation of INHBA, CYP11A1, CYP19A1, and LHCGR. TSZF also enhanced oocyte FIGLA expression, indicating improved GC-oocyte communication. Moreover, TSZF increased the survival rate and promoted the growth of primordial follicles isolated both in vivo and in vitro. The active components of TSZF, namely hyperoside, chlorogenic acid, isoquercitrin and astragalin, can reverse the decrease in viability of TG-induced KGN cells and effectively bind to INHBA and FIGLA.

CONCLUSION: Our data demonstrate that TSZF and its active components mitigate TG-induced ovarian injury through modulation of the ovarian microenvironment, preservation of the primordial follicle pool, promotion of GC differentiation, and enhancement of GC-oocyte communication, with INHBA identified as a key regulatory node. These findings support a protective and recovery‑associated role of TSZF in POI.