Protease-Resistant Diabetes Mellitus Placenta with Advanced Glycation End-Products Accumulation Monitored by Scanning Acoustic Microscopy

Background: Pregnancy complicated by diabetes mellitus (DM) leads to reduced placental blood flow, impacting both maternal and fetal health. Although DM placentas exhibit distinct histological changes, clinical glycemic control has failed to reverse these structural alterations. Hyperglycemia induces protein glycation and advanced glycation end-products (AGEs) accumulation, which are highly resistant to degradation. This study aimed to evaluate the structural and functional changes of DM placentas by monitoring their enzymatic degradation process using scanning acoustic microscopy (SAM) and comparing the findings with AGEs immunohistochemistry. Methods: Placental tissues from 6 DM and 5 non-DM pregnancies were fixed, embedded in paraffin, and sectioned. The sections were subjected to time-course proteolytic digestion using actinase E (pronase E) at 37 ?C. At designated time points, the sections were analyzed using a high-frequency (320 MHz) SAM system to measure sound attenuation (SA) and speed of sound (SOS), generating acoustic histological images. Corresponding or adjacent sections underwent routine light microscopy (LM) with hematoxylin and eosin staining, as well as immunohistochemical staining using anti-AGEs antibodies (anti-AKR 1B1 and anti-N?-carboxymethyl lysine). Acoustic values across different tissue components and time points were statistically compared. Results: The high-resolution SAM system successfully captured chronological changes in SA and SOS images during enzymatic digestion, reflecting structural degradation. DM placentas exhibited significantly higher resistance to protease degradation over time compared to non-DM placentas. This protease resistance strongly correlated with the localized accumulation and intensity of AGEs immunoreactivity observed via LM. Conclusion: SAM combined with enzymatic digestion effectively visualizes the microstructural and functional differences between DM and non-DM placentas. The accumulation of degradation-resistant AGEs likely underlies the irreversible structural alterations in DM placentas, offering novel insights into placental pathology beyond conventional optical microscopy.