**4.1 Innovative methods to objectivate premature cervical microstructure remodeling**

Non-invasive methods are adopted in maternal-fetal medicine for collagen architecture assessment, clinicians collaborating with biomechanical engineers and other scientists to reveal crucial structural properties changes during pregnancy and labor. Cervical microstructure is a very good objective for new proposed *in vivo* techniques based on quantification of mechanical, optical, or electrical tissue properties: increased hydration with preconceptional collagen organization loss, and/or cervical elasticity [95].

Histopathology is the golden standard for tissue remodeling assessment. Electron/polarized light microscopy, two-photon microscopy, and fluorescence microscopy, with different dyes/antibodies for staining and with limited physicochemical changes on biological tissue, are in trend for slice sizes and collagen content accurate diagnosis. SHG scanning endomicroscopy was proposed to visualize gestational CR in many studies in Europe and the USA. There were recently three accurate collagen fiber extraction techniques (in 3D volume) for quantitative collagen content assessment [96]. SHG permits cervical collagen content longitudinal assessments along pregnancy [12 (first trimester), 23 (second trimester), and 32 (third trimester) gestational weeks], with images archive, and comparison at each visit [97].

Literature mentions numerous new approaches, depending on sites/centers resources and medical staff training, to visualize cervix microstructure: near-infrared spectroscopy and near-infrared Raman microspectroscopy (optical techniques), light-induced fluorescence by callascope—specifically designed to assess cervical ripening, and functions by measuring the natural fluorescence of cervical non-soluble collagen, other methods combine ultrasound to new capabilities to visualize cervix deformability at different levels, by devices intravaginal/intracervical introduced, capturing differences between stiff and soft cervix, as aspiration and cervical consistency index, spectroscopic photo-acoustic imaging (first used in USA, for collagen/ water ratio assessment in murine cervices [88], acoustic attenuation; all these methods are not in this subchapter focus.

### **4.2 Ultrasound-based techniques for cervical microstructure evaluation**

Different from conventional ultrasound, various imaging modalities are actually prone to visualize changes in reproductive organs as collagen fibers content and characteristics (density, size, morphology, and orientation/alignment) along pregnancy, and associated to computational tools may quantify, and predict softness abnormal progress, and shift to ripening, before cervical shortness and funneling, in asymptomatic cases, for early sPTB prevention/delay.
