Birth mode is associated with layer-specific mechanical changes in fetal membranes.

Rupture of fetal membranes and subsequent full-term birth are prerequisites for neonatal health, and a preterm rupture can lead to life-threatening complications. Our study determines the mechanical properties of term fetal membranes to identify perinatal structural changes by a unique biophysical multiscale approach, including atomic force microscopy, shear rheology, tabletop magnetic resonance elastography (MRE), and high-resolution optical microscopy. Fetal membranes from term spontaneous vaginal deliveries were compared to those from primary cesarean sections, used as a control group for pre-labor membranes. Spontaneously delivered term fetal membranes are softer and easier to deform in MRE experiments (median stiffness: 1.9 kPa, IQR 1.6-2.4) compared to controls (4.7 kPa, IQR 3.8-5.6); p < 0.001) and show increased water diffusion (median: 1.78 × 10-3 mm2/s, IQR: (1.65-1.84) × 10-3 vs. 1.66 × 10-3 mm2/s, IQR (1.60-1.73) × 10-3; p = 0.047). Their intermediate connective tissue layer (i.e. the collagen-rich area enclosed by the amnion and chorion) exhibits less ordered fiber alignment (median order parameter: 0.52, IQR 0.44-0.58 vs. 0.55, IQR 0.47-0.62; p = 0.04) and a looser fiber structure, as indicated by a significantly lower fiber area fraction (median: 0.33, IQR 0.25-0.46 vs. 0.73, IQR 0.63-0.88; p < 0.001) compared to the control membranes. These layer-specific changes in both structure and viscoelasticity are evidence for the dominant role of the intermediate connective tissue in maintaining membrane stability and the onset of rupture. Our mechanical and histopathological findings highlight the potential of mechanics-based screening-methods to assess the risk of preterm rupture and preterm birth to reduce neonatal morbidity.