Mueller matrix imaging for collagen scoring in mice model of pregnancy

Preterm birth risk is associated with early softening of the uterine cervix in pregnancy due to the accelerated remodeling of collagen extracellular matrix. Studies of mice model of pregnancy were performed with an imaging Mueller polarimeter at different time points of pregnancy to find polarimetric parameters for collagen scoring. Mueller matrix images of the unstained sections of mice uterine cervices were taken at day 6 and day 18 of 19-days gestation period and at different spatial locations through the cervices. The logarithmic decomposition of the recorded Mueller matrices mapped the depolarization, linear retardance, and azimuth of the optical axis of cervical tissue. These images highlighted both the inner structure of cervix and the arrangement of cervical collagen fibers confirmed by the second harmonic generation microscopy. The statistical analysis and two-Gaussians fit of the distributions of linear retardance and linear depolarization in the entire images of cervical tissue (without manual selection of the specific regions of interest) quantified the randomization of collagen fibers alignment with gestation time. At day 18 the remodeling of cervical extracellular matrix of collagen was measurable at the external cervical os that is available for the direct optical observations in vivo. It supports the assumption that imaging Mueller polarimetry holds promise for the fast and accurate collagen scoring in pregnancy and the assessment of the preterm birth risk

Quantitative Evaluation of Collagen Crosslinks and Corresponding Tensile Mechanical Properties in Mouse Cervical Tissue during Normal Pregnancy

The changes in the mechanical integrity of the cervix during pregnancy have implications for a successful delivery. Cervical collagens are known to remodel extensively in mice with progressing gestation leading to a soft cervix at term. During this process, mature crosslinked collagens are hypothesized to be replaced with immature less crosslinked collagens to facilitate cervical softening and ripening. To determine the mechanical role of collagen crosslinks during normal mouse cervical remodeling, tensile load-to-break tests were conducted for the following time points: nonpregnant (NP), gestation day (d) 6, 12, 15, 18 and 24 hr postpartum (PP) of the 19-day gestation period. Immature crosslinks (HLNL and DHLNL) and mature crosslinks (DPD and PYD) were measured using ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS). There were no significant changes in the total immature crosslink density (HLNL+DHLNL mol per collagen mol) throughout normal mouse gestation (range: 0.31–0.49). Total mature crosslink density (PYD+DPD mol per collagen mol) decreased significantly in early softening from d6 to d15 (d6: 0.17, d12: 0.097, d15: 0.026) and did not decrease with further gestation. The maturity ratio (total mature to total immature crosslinks) significantly decreased in early softening from d6 to d15 (d6: 0.2, d15: 0.074). All of the measured crosslinks correlated significantly with a measure of tissue stiffness and strength, with the exception of the immature crosslink HLNL. This data provides quantitative evidence to support the hypothesis that as mature crosslinked collagens decline, they are replaced by immature collagens to facilitate increased tissue compliance in the early softening period from d6 to d15

Protocol to dissociate epithelia from non-pregnant and pregnant mouse cervical tissue for single-cell RNA-sequencing

Summary: A challenge in studying cervical epithelial cell biology at the single-cell level is that differentiated subtypes, in particular mucus-secreting goblet cells, are sensitive to disassociating enzymes making isolation of all epithelial subpopulations difficult. Here we present a protocol to dissociate epithelia from non-pregnant and pregnant mouse cervical tissue for single-cell RNA-sequencing. We describe steps for harvesting cervices, preparing cervical tissue, dissociation of cervical cells, and viability checks. We then detail library preparation, sequencing, and procedure for data analysis.For complete details on the use and execution of this protocol, please refer to Cooley et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Changes of Large Molecular Weight Hyaluronan and Versican in the Mouse Pubic Symphysis Through Pregnancy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)During pregnancy, the mouse pubic symphysis undergoes expansion and remodeling resulting in formation of a flexible and elastic interpubic ligament allowing passage of a term fetus. In the current study, we sought to identify and characterize components of the extracellular matrix that likely play an important role in elongation and flexibility of the interpubic ligament during parturition. Mouse pubic symphyses and interpubic ligaments collected at time points during pregnancy and postpartum were utilized to evaluate collagen type, collagen content, processing and solubility, matricellular protein, and proteoglycan expression and quantitative assessment of all glycosaminoglycans. These studies revealed increased gene expression for hyaluronan synthase 1, hyaluronan synthase 2, and versican on Gestation Day 18 as well as a decline in protein expression for the versican-degrading protease a disintegrin-like and metalloprotease with thrombospondin type 1 (ADAMTS1) motif. These findings suggest that the primary mediators of increased elongation and flexibility of the interpubic ligament at term result from increased synthesis and reduced metabolism of viscoelasticity-promoting molecules such as high molecular weight hyaluronan and versican.862Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)U.S. National Institutes of Health [P01 HD011149]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Cervical Softening During Pregnancy: Regulated Changes in Collagen Cross-Linking and Composition of Matricellular Proteins in the Mouse1

A greater understanding of the parturition process is essential in the prevention of preterm birth, which occurs in 12.7% of infants born in the United States annually. Cervical remodeling is a critical component of this process. Beginning early in pregnancy, remodeling requires cumulative, progressive changes in the cervical extracellular matrix (ECM) that result in reorganization of collagen fibril structure with a gradual loss of tensile strength. In the current study, we undertook a detailed biochemical analysis of factors in the cervix that modulate collagen structure during early mouse pregnancy, including expression of proteins involved in processing of procollagen, assembly of collagen fibrils, cross-link formation, and deposition of collagen in the ECM. Changes in these factors correlated with changes in the types of collagen cross-links formed and packing of collagen fibrils as measured by electron microscopy. Early in pregnancy there is a decline in expression of two matricellular proteins, thrombospondin 2 and tenascin C, as well as a decline in expression of lysyl hydroxylase, which is involved in cross-link formation. These changes are accompanied by a decline in both HP and LP cross-links by gestation Days 12 and 14, respectively, as well as a progressive increase in collagen fibril diameter. In contrast, collagen abundance remains constant over the course of pregnancy. We conclude that early changes in tensile strength during cervical softening result in part from changes in the number and type of collagen cross-links and are associated with a decline in expression of two matricellular proteins thrombospondin 2 and tenascin C

Second harmonic generation imaging as a potential tool for staging pregnancy and predicting preterm birth

We use second harmonic generation (SHG) microscopy to assess changes in collagen structure of murine cervix during cervical remodeling of normal pregnancy and in a preterm birth model. Visual inspection of SHG images revealed substantial changes in collagen morphology throughout normal gestation. SHG images collected in both the forward and backward directions were analyzed quantitatively for changes in overall mean intensity, forward to backward intensity ratio, collagen fiber size, and porosity. Changes in mean SHG intensity and intensity ratio take place in early pregnancy, suggesting that submicroscopic changes in collagen fibril size and arrangement occur before macroscopic changes become evident. Fiber size progressively increased from early to late pregnancy, while pores between collagen fibers became larger and farther apart. Analysis of collagen features in premature cervical remodeling show that changes in collagen structure are dissimilar from normal remodeling. The ability to quantify multiple morphological features of collagen that characterize normal cervical remodeling and distinguish abnormal remodeling in preterm birth models supports future studies aimed at development of SHG endoscopic devices for clinical assessment of collagen changes during pregnancy in women and for predicting risk of preterm labor which occurs in 12.5% of all pregnancies