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

Abstract

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.

In vivo Raman spectroscopy for biochemical monitoring of the human cervix throughout pregnancy

“…33 Decreases in ECM peaks are explained by the process of ECM remodeling where collagen becomes increasingly soluble due to reduced cross-links and dispersed at a lower concentration. 4,15 Trivalent collagen cross-links have been observed at 1660 cm −1 , and reductions in these cross-links (which have been previously reported in women 27 and mice 27,52 ) likely contribute to the observed decrease in this peak during pregnancy. 53 Furthermore, the 1657 cm −1 peak (also known as the amide I region) broadened in preparation for delivery, a phenomenon that is known to be caused by structural disorder such as collagen fiber dispersion.…”

The mechanical role of the cervix in pregnancy

“…Cervical remodeling during pregnancy has been measured mainly in rodent tissue and in some human tissue studies (Akins et al, 2011; Danforth, 1983, 1947; Danforth et al, 1960, 1974; Holt et al, 2011; Leppert, 1992, 1995, 1998; Timmons et al, 2010b; Word et al, 2007; Yoshida et al, 2014a,b), where cervical softening during pregnancy is shown to depend on alterations in the collagen fiber organization, collagen fiber crosslinking, and the GAG and water content. In our preliminary collagen studies, we find evidence of collagen remodeling during pregnancy at multiple hierarchal length-scales of the collagen fiber network.…”

“…In our preliminary collagen studies, we find evidence of collagen remodeling during pregnancy at multiple hierarchal length-scales of the collagen fiber network. Interestingly, the total collagen content per dry weight is thought to not change throughout remodeling in both humans (Myers et al, 2009) and mice (Akins et al, 2011; Yoshida et al, 2014a). Instead, pregnant human tissue is more soluble in weak acids indicating a reduction of collagen crosslinks (Myers et al, 2009).…”

“…Instead, pregnant human tissue is more soluble in weak acids indicating a reduction of collagen crosslinks (Myers et al, 2009). In mouse cervical tissue, there is a significant decrease in mature crosslinks (pyridinoline [PYD] and deoxypyridinoline [DPD]) during the cervical softening phase in early pregnancy (Yoshida et al, 2014a). This collagen crosslink decrease is accompanied by a decrease in the structural stiffness of whole tissue specimens.…”

Changes in cervical stiffness during pregnancy: Preliminary assessment with shear wave elasticity imaging in the rhesus macaque

“…This can result in interand intra-subject variability of SWS estimates. Given that tissue strength depends upon the degree of collagen crosslinking, [30,31] this complicated structure is expected to disrupt close to term to allow the cervix to soften. Thus, we hypothesize that the reduction in variance close to term could be explained by disruption of collagen crosslinks and the consequent structural homogenization.…”