Validation of ultrasound biomicroscopy for the assessment of xenogeneic testis tissue grafts and cell implants in recipient mice

Background: Subcutaneous grafting/implantation of neonatal testis tissue/cells from diverse donor species into recipient mice can be used as an in vivo model to study testis development, spermatogenesis, and steroidogenesis. Ultrasound biomicros- copy (UBM) allows obtaining high definition cross-sectional images of tissues at mi- croscopic resolutions. Objectives: The present study was designed to (a) validate the use of UBM for non- invasive monitoring of grafts/implants overtime and to (b) correlate UBM findings with the morphological attributes of recovered grafts/implants. 3 Materials and methods: Testis tissue fragments (~14 mm , each) and cell aggregates (100 × 106 cells, each) obtained from 1-week-old donor piglets (n = 30) were grafted/ implanted under the back skin of immunodeficient mice (n = 6) in eight analogous sites per mouse. Three-dimensional transcutaneous Doppler UBM was performed, and a randomly selected graft and its corresponding implant were recovered at 2, 4, 6, and 8 weeks. Results: Graft/implant weight (P = .04) and physical height (P = .03) increased overtime. The dynamics of physical length and volume increases over time differed between tissue grafts and cell implants (P = .02 and 0.01 for sample type*time interactions, re- spectively). UBM-estimated volume was correlated with the post-recovery weight and volume of the grafts/implants (r = 0.98 and r = 0.99, respectively; P < .001). Pre- and post-recovery length and height of the grafts/implants were positively and strongly correlated (r = 0.50, P = .01; r = 0.70, P = .001) and so were the areas covered by cordal, non-cordal, or fluid-filled cavities between UBM and histology (r = 0.87, P < .001). Discussion and conclusion: UBM findings correlated with physical attributes of the grafts/implants, validating its use as a non-invasive high-fidelity tool to quantify the developmental changes in ectopic testis tissue grafts and cell implants, potentially leading to a reduction in the number of recipient mice needed for similar experiments.