Kyoko Yoshida

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.

Measuring the compressive viscoelastic mechanical properties of human cervical tissue using indentation

The human cervix is an important mechanical barrier in pregnancy which must withstand the compressive and tensile forces generated from the growing fetus. Premature cervical shortening resulting from premature cervical remodeling and alterations of cervical material properties are known to increase a woman׳s risk of preterm birth (PTB). To understand the mechanical role of the cervix during pregnancy and to potentially develop indentation techniques for in vivo diagnostics to identify women who are at risk for premature cervical remodeling and thus preterm birth, we developed a spherical indentation technique to measure the time-dependent material properties of human cervical tissue taken from patients undergoing hysterectomy. In this study we present an inverse finite element analysis (IFEA) that optimizes material parameters of a viscoelastic material model to fit the stress-relaxation response of excised tissue slices to spherical indentation. Here we detail our IFEA methodology, report compressive viscoelastic material parameters for cervical tissue slices from nonpregnant (NP) and pregnant (PG) hysterectomy patients, and report slice-by-slice data for whole cervical tissue specimens. The material parameters reported here for human cervical tissue can be used to model the compressive time-dependent behavior of the tissue within a small strain regime of 25%.

A continuous fiber distribution material model for human cervical tissue

The uterine cervix during pregnancy is the vital mechanical barrier which resists compressive and tensile loads generated from a growing fetus. Premature cervical remodeling and softening is hypothesized to result in the shortening of the cervix, which is known to increase a woman׳s risk of preterm birth. To understand the role of cervical material properties in preventing preterm birth, we derive a cervical material model based on previous mechanical, biochemical and histological experiments conducted on nonpregnant and pregnant human hysterectomy cervical tissue samples. In this study we present a three-dimensional fiber composite model that captures the equilibrium material behavior of the tissue in tension and compression. Cervical tissue is modeled as a fibrous composite material, where a single family of preferentially aligned and continuously distributed collagen fibers are embedded in a compressible neo-Hookean ground substance. The total stress in the collagen solid network is calculated by integrating the fiber stresses. The shape of the fiber distribution is described by an ellipsoid where semi-principal axis lengths are fit to optical coherence tomography measurements. The composite material model is fit to averaged mechanical testing data from uni-axial compression and tension experiments, and averaged material parameters are reported for nonpregnant and term pregnant human cervical tissue. The model is then evaluated by investigating the stress and strain state of a uniform thick-walled cylinder under a compressive stress with collagen fibers preferentially aligned in the circumferential direction. This material modeling framework for the equilibrium behavior of human cervical tissue serves as a basis to determine the role of preferentially-aligned cervical collagen fibers in preventing cervical deformation during pregnancy.

Cervical collagen network remodeling in normal pregnancy and disrupted parturition in Antxr2 deficient mice.

The remodeling of the cervix from a rigid barrier into a compliant structure, which dilates to allow for delivery, is a critical process for a successful pregnancy. Changes in the mechanical properties of cervical tissue during remodeling are hypothesized to be related to the types of collagen crosslinks within the tissue. To further understand normal and abnormal cervical remodeling, we quantify the material properties and collagen crosslink density of cervical tissue throughout pregnancy from normal wild-type and Anthrax Toxin Receptor 2 knockout (Antxr2-/-) mice. Antxr2-/- females are known to have a parturition defect, in part, due to an excessive accumulation of extracellular matrix proteins in the cervix, particularly collagen. In this study, we determined the mechanical properties in gestation-timed cervical samples by osmotic loading and measured the density of mature collagen crosslink, pyridinoline (PYD), by liquid chromatography tandem mass spectrometry (LC-MSMS). The equilibrium material response of the tissue to loading was investigated using a hyperelastic material model where the stresses in the material are balanced by the osmotic swelling tendencies of the glycosaminoglycans and the tensile restoring forces of a randomly-oriented crosslinked collagen fiber network. This study shows that the swelling response of the cervical tissue increased with decreasing PYD density in normal remodeling. In the Antxr2-/- mice, there was no significant increase in swelling volume or significant decrease in crosslink density with advancing gestation. By comparing the ECM-mechanical response relationships in normal and disrupted parturition mouse models this study shows that a reduction of collagen crosslink density is related to cervical softening and contributes to the cervical remodeling process.

Material properties of mouse cervical tissue in normal gestation.

UNLABELLED An appropriately timed cervical remodeling process is critical for a healthy delivery, yet little is known about the material property changes of the cervix in pregnancy because obtaining human tissue samples is difficult. Rodent models offer advantages including accurately timed pregnant tissues and genetically altered models. Determining the material properties of the mouse cervix, however, is challenging because of its small size and complex geometry. The aim of this study is to quantify cervical material property changes in a normal mouse pregnancy using a microstructurally-inspired porous fiber composite model. We mechanically test intact, whole, gestation-timed mouse cervix by pulling apart tensioned sutures through its inner canal. To interpret our mechanical testing results, we conduct an inverse finite element analysis, taking into account the combined loading state of the thick-walled cylindrical tissue. We fit the material model to previous osmotic swelling data and load-deformation data from this study using a nonlinear optimization scheme, and validate the model by predicting a separate set of deformation data. Overall, the proposed porous fiber composite model captures the mechanical behavior of the mouse cervix in large deformation. The evolution of cervical material parameters indicates that in a normal mouse pregnancy, the cervix begins to soften between day 6 and day 12 of a 19-day gestation period. The material parameter associated with the collagen fiber stiffness decreases from 3.4MPa at gestation day 6 to 9.7e-4MPa at gestation day 18, while the ground substance stiffness decreases from 2.6e-1MPa to 7.0e-4MPa. STATEMENT OF SIGNIFICANCE Accelerated cervical remodeling can lead to extremely premature births. Little is known, however, about the material property changes of the cervix in pregnancy because pregnant human tissue samples are limited. Rodent models overcome this limitation and provide access to gestation-timed samples. Measuring the material property changes of the mouse cervix in pregnancy is challenging due to its small size and complex geometry. Here, we establish a combined experimental and modeling framework. We use this framework to determine the cervical material property changes throughout a normal mouse pregnancy. We present our experimental methods for mechanically testing whole, intact cervical tissue samples. We fit a porous fiber composite material model to the mechanical data and show that the mouse cervix begins to soften between day 6 and day 12 of a 19-day gestation period.

Direct Measurement of the Permeability of Human Cervical Tissue

The mechanical integrity of the uterine cervix is critical for a pregnancy to successfully reach full term. It must be strong to retain the fetus throughout gestation and then undergo a remodeling and softening process before labor for delivery of the fetus. It is believed that cervical insufficiency (CI), a condition in pregnancy resulting in preterm birth (PTB), is related to a cervix with compromised mechanical strength which cannot resist deformation caused by external forces generated by the growing fetus. Such PTBs are responsible for infant developmental problems and in severe cases infant mortality. To understand the etiologies of CI, our overall research goal is to investigate the mechanical behavior of the cervix. Permeability is a mechanical property of hydrated collagenous tissues that dictates the time-dependent response of the tissue to mechanical loading. The goal of this study was to design a novel soft tissue permeability testing device and to present direct hydraulic permeability measurements of excised nonpregnant (NP) and pregnant (PG) human cervical tissue from women with different obstetric histories. Results of hydraulic permeability testing indicate repeatability for specimens from single patients, with an order of magnitude separating the NP and PG group means (2.1 ± 1.4×10(-14) and 3.2 ± 4.8×10(-13)m(4)/N[middle dot]s, respectively), and large variability within the NP and PG sample groups. Differences were found between samples with similar obstetric histories, supporting the view that medical history may not be a good predictor of permeability (and therefore mechanical behavior) and highlighting the need for patient-specific measurements of cervical mechanical properties. The permeability measurements from this study will be used in future work to model the constitutive material behavior of cervical tissue and to develop in vivo diagnostic tools to stage the progression of labor.

Steroid hormones are key modulators of tissue mechanical function via regulation of collagen and elastic fibers

The extracellular matrix (ECM) plays an active and dynamic role that both reflects and facilitates the functional requirements of a tissue. The mature ECM of the nonpregnant cervix is drastically reorganized during pregnancy to drive changes in tissue mechanics that ensure safe birth. In this study, our research on mice deficient in the proteoglycan decorin have led to the finding that progesterone and estrogen play distinct and complementary roles to orchestrate structural reorganization of both collagen and elastic fibers in the cervix during pregnancy. Abnormalities in collagen and elastic fiber structure and tissue mechanical function evident in the cervix of nonpregnant and early pregnant decorin-null mice transiently recover for the remainder of pregnancy only to return 1 month postpartum. Consistent with the hypothesis that pregnancy levels of progesterone and estrogen may regulate ECM organization and turnover, expressions of factors required for assembly and synthesis of collagen and elastic fibers are temporally regulated, and the ultrastructure of collagen fibrils and elastic fibers is markedly altered during pregnancy in wild-type mice. Finally, utilizing ovariectomized nonpregnant decorin-null mice, we demonstrate structural resolution of collagen and elastic fibers by progesterone or estrogen, respectively, and the potential for both ECM proteins to contribute to mechanical function. These investigations advance understanding of regulatory factors that drive specialized ECM organization and contribute to an understanding of the cervical remodeling process, which may provide insight into potential complications associated with preterm birth that impact 9.6% of live births in the United States.

Time-Dependent Indentation Response of Human Cervical Tissue

More than half a million babies are born prematurely in the United States, and the rate of preterm birth (PTB) is on the rise [1]. Infants born prematurely account for a high percentage of perinatal mortalities, and preterm infants that survive are at risk for long-term morbidities including neurologic, respiratory, cardiovascular and gastrointestinal complications [2]. Altered mechanical and biochemical properties of the uterine cervix are suspected to cause premature and spontaneous cervical dilation, which is associated with a leading cause of PTB known as cervical insufficiency (CI). The impact of this condition is unknown because diagnosing CI remains controversial. Multiple etiologies are believed to lead to an insufficient cervix, or a soft cervix. Yet, there is no standard method to quantify the mechanical strength of the cervix, limiting the ability to discern these etiologies, to stage the progression of labor, and to identify and manage high-risk CI patients.© 2012 ASME

Inverse Finite Element Analysis of the Indentation Response of Human Cervical Tissue

The mechanical function of the cervix is crucial during pregnancy when it is required to resist the compressive and tensile forces generated from the growing fetus. Pathologies of the cervical extracellular matrix (ECM), premature cervical remodeling, and alterations of cervical material properties have been implicated in placing women at high-risk for preterm birth (PTB). To understand the mechanical role of the cervix during pregnancy and to potentially identify etiologies for PTB, the overall goal of our group is to quantify ECM-material property relationships in normal and diseased human cervical tissue. In this study we present an inverse finite element analysis (IFEA) that optimizes material parameters of a viscoelastic material model to fit the stress-relaxation response of excised tissue slices to spherical indentation. Here we detail our IFEA methodology, report viscoelastic material parameters for cervical tissue slices from nonpregnant (NP) and pregnant (PG) hysterectomy patients, and report slice-by-slice data for whole cervical tissue specimens.Copyright

Cervical Collagen Network Remodeling in Normal and Disrupted Parturition Mouse Models

The cervix plays an important role in pregnancy as a mechanical barrier to prevent preterm birth (PTB). The material strength of the cervix can be attributed to its extracellular matrix (ECM), a network of cross-linked collagens (types I and III) embedded within a viscous matrix of glycosaminoglycans (GAG). GAGs are negatively charged polysaccharides that provide a fixed charge density (FCD) within the tissue to maintain hydration. Throughout gestation, the ECM of the cervix undergoes a remodeling process characterized by three stages: gradual softening in early pregnancy, a rapid increase in tissue distensibility termed ripening in late pregnancy, and repair at post partum1. As the cervix softens and ripens, mature collagen cross-links break down while GAG content increases2,3. Previous research has shown that these changes in collagen and GAGs correlate to a mechanically softer cervix at term4.© 2013 ASME