Superovulation-induced changes in murine ovarian perifollicular angiogenesis

In brief Superovulation is widely used in reproductive technologies, but its impact on ovarian vascular development and oocyte quality remains poorly understood. This study provides the first 3D quantitative analysis of perifollicular vasculature in superovulated mice, revealing dose-dependent vascular changes and offering a novel imaging approach for reproductive research. Abstract Superovulation is widely used to maximise oocyte/embryo yield in animal models. However, it has been implicated in disrupting normal follicular development, potentially affecting perifollicular angiogenesis. This study investigated the impact of superovulation on ovarian perifollicular neoangiogenesis using light sheet fluorescence microscopy (LSFM), and by quantitatively profiling the three-dimensional (3D) perifollicular capillary bed in murine antral follicles. Dioestrus CD1 mice received 2.5, 5.0 or 7.5 IU pregnant mare serum gonadotrophin (PMSG) intraperitoneally, and ovaries were collected 24 and 48 h later, with those from normal cycling females (dioestrus, proestrus or oestrus) as controls. Ovaries were fixed and labelled with fluorescently tagged wheat germ agglutinin lectin and anti-CD34 to visualise the oocyte zona pellucida and thecal vasculature, respectively. Optically cleared samples were imaged using LSFM, and 3D volume rendering, vessel segmentation and image analysis were performed using Arivis Vision 4D and Fiji. Quantitative metrics including vessel volume, length, branching, density, spatial arrangement and oocyte characteristics were profiled. Statistical analysis was based on Kruskal–Wallis tests. PMSG-induced superovulation showed dose-dependent effects on perifollicular vasculature, causing premature (24 h) neoangiogenesis at 7.5 IU (increase in total vessel volume, length and number of branches, and decrease in average branch length; P < 0.05 for all), and reduced final (48 h) vessel density at 2.5 IU compared to naturally cycling animals ( P < 0.05). The early angiogenesis observed at 7.5 IU may reflect a compensatory mechanism, while the reduced density at 2.5 IU suggests an insufficient angiogenic response. By contrast, intrafollicular metrics were largely unaffected. This study provides the first comprehensive quantitative 3D analysis of thecal vasculature and oocytes in murine ovaries, and highlights its potential applications in other areas of reproductive biology.