Biomechanical and Microstructural Characterization of Postpartum Uterine Involution in the Mouse

Introduction: Uterine involution is the natural biological process through which the pregnant uterus returns to its pre-pregnant state after delivery. Elastin and collagen within the uterine wall increase during pregnancy and are reabsorbed and reconstructed throughout involution as the uterus shrinks toward its original size. This process is marked by enzymatic and growth factor activity – such as MMPs, TIMPs, and IGF – suggesting there is involution-induced remodeling of the uterine extracellular matrix (ECM) after birth. Together, there is a relationship between pregnancy, postpartum (PP) involution, and ECM remodeling that is not well understood.
Millions of women give birth every year and incomplete or dysregulated uterine involution is associated with several pregnancy-related complications, including PP hemorrhage and uterine atony. Despite the prevalence of birth-related complications, to date there has been limited work characterizing the biomechanical changes associated with PP uterine involution. During involution, the ECM is degraded and reorganized in a reverse remodeling process that is unique to reproductive tissue, making the postpartum uterus an ideal system for studying ECM remodeling.
To this end, we measured time-dependent changes in uterine biomechanics using a mouse model of PP involution. These findings were then correlated with microstructural changes (e.g., elastin and collagen networks) to investigate ECM remodeling and its impact on uterine tissue mechanics. Understanding these concepts will help to identify the biological mechanisms driving postpartum healing and ECM remodeling dynamics in the uterus, which has potential implications in both regenerative medicine and tissue engineering applications.

Materials and

Methods: Uterine tissue was collected from adult female mice at 1, 3, 7, and 14 days following vaginal delivery (n=3-6 per time). Nulliparous mice were used as controls. Prior to testing and analysis, uterine tissue was frozen in DMEM containing 10% DMSO using a controlled-rate freezing vessel.
To investigate the biomechanical properties of the PP uterus, an established biaxial extension-inflation technique was employed. Uterine tissue was thawed and cannulated onto 17G-21G blunt needles based on diameter. After preconditioning, the uterus was first pressurized from 5 to 80 mmHg while at the in vivo length and ±5% the in vivo length (as estimated by the axial force-pressure relationship) followed by axial stretching up to 5 g of axial force at fixed pressures of 5, 25, 55, and 80 mmHg. Outer diameter, pressure, axial stretch, and axial force were recorded throughout the test and biaxial stress-stretch curves were developed. After biaxial testing, the uterus was pressurized to failure. Unloaded thickness was measured via Optical Coherence Tomography (OCT) imaging of uterine rings.
For microstructural analysis, untested uterine segments were embedded in OCT, cryosectioned at 8 µm, and stained with either an immunofluorescence protocol targeting elastin and α-smooth muscle actin (αSMA) or a Picrosirius red (PSR) staining protocol to visualize collagen. Slides were imaged and quantified using FIJI and custom Matlab scripts.
For statistical analysis, normality and homogeneity of variance was determined and appropriate comparison testing and post-hoc analysis (one-way ANOVA with Tukey HSD or Kruskal-Wallis with Dunn’s) was performed in GraphPad Prism.

Results, Conclusions, and Discussions: Unloaded uterine diameter and wall thickness are increased immediately following birth and decrease throughout PP involution before returning to pre-pregnant levels by 14dPP (Fig.1 A,B). Biaxial testing revealed dynamic changes in the outer diameter (Fig.1 C, top) and axial stretch (Fig.1 C, bottom). Despite large changes in diameter, the circumferential stresses were similar between controls and all time points (Fig.1E, top). However, at 1dPP there is a leftward shift of the axial stress-stretch curve, indicating a structural stiffening that resolves and resembles the behavior of the control uterus throughout involution (Fig.1E, bottom). The pressure necessary to induce rupture in the PP uterus is decreased at all PP time points, relative to control, (Fig.1D) indicating potential pregnancy-induced changes in uterine integrity that do not resolve after involution despite similar geometries.
Dynamic ECM remodeling of the PP uterine wall was examined histologically. The normalized area of elastin and αSMA identified via immunofluorescence staining was nearly constant throughout involution (Fig.2 A,B). While PSR staining demonstrated that the majority of uterine collagen is highly organized (red/orange), the proportion of disorganized collagen (yellow/green) was decreased at 3dPP (Fig.2 A,D). This was consistent with an increase in myometrium thickness (from αSMA staining) at 3dPP that subsequently decreased at later involution times. The percentage of myometrial elastin gradually increased throughout involution and remained elevated, relative to control, at the latest timepoint of 14dPP. Within the myometrium, the percentage of αSMA+ area was decreased early and remained lower than control at 14dPP despite increasing relative to the lowest expression at 3dPP (Fig 2 A,C). These changes in the myometrium, compared to the overall uterine wall structure, indicate that the myometrium experiences substantial ECM remodeling throughout involution. That said, at 14dPP the myometrium remains different than control, suggesting there may be incomplete uterine remodeling at this stage in the involution process.
In the future, this work will expand upon the current cohort of animals by including longer-term time points to further investigate pregnancy-induced changes and PP remodeling of the uterus. Characterization of the underlying mechanobiology of uterine involution can provide targets for developing therapeutic strategies to promote postpartum healing.

Acknowledgements (Optional): This work is supported by internal seed grant funds provided by the Washington University in St. Louis Center for Regenerative Medicine and the Center for Women's Health Engineering.