Spatially Mapping the Mechanical and Structural Properties of the Uterine Fibroid-Myometrium Interface

Introduction: Uterine fibroids, also known as leiomyomas, are defined as the non-cancerous growth of tumors in the uterus and affect nearly 70−80% of women by age 50 [1−2]. Uterine fibroids are a common cause of heavy menstrual bleeding, pelvic pain, infertility, and preterm birth, and account for more than 200,000 hysterectomies performed in the U.S. annually [2−3]. While fibroids have been documented to grow as large as 30 cm in diameter, all originate initially as seedling fibroids on the mm scale [4]. It is well established that uterine fibroids are significantly stiffer than the surrounding myometrium tissue using bulk tissue testing approaches, exhibiting both intra- and inter-patient variability [5−9]. However, the spatial variation of material properties at the fibroid-myometrium interface is currently unknown. Such knowledge is critical for the development of novel in vivo tools for the detection, diagnosis, and treatment of uterine fibroids. Therefore, this study aims to spatially map the time-dependent material properties of seedling uterine fibroids and their surrounding myometrium at the microscale.

Materials and

Methods: In accordance with IRB approval at Columbia University Irving Medical Center (CUIMC), human uterine tissues embedded with seedling fibroids were collected from nonpregnant women (N=13), aged 18−50, undergoing total hysterectomies for noncancerous indications. From the uterine tissue collected, 48 mm-sized fibroids were identified based on gross, visual characteristics and manual palpation. The material properties of the fibroid-myometrium interface were subsequently characterized with nanoindentation testing as a rectangular region. As shown in Fig. 1B, the length (𝑋) of the tested region was equivalent to the full length of the fibroid (𝐹L) plus a fixed 3 mm of adjacent myometrium tissue. The width (𝑌) of the tested region was centered relative to the fibroid’s width (𝐹w) and equivalent to 0.4*𝐹w. The distance between indentation points was fixed at 200μm. Distant myometrium tissue was also tested and was defined as a region of myometrium greater than 5 mm from any visible fibroid. Using a 50 µm probe radius, samples were indented to a fixed depth of 4µm for 15s, corresponding to a 5% strain, to yield a load relaxation curve approaching equilibrium (Fig. 1C). Data was fit with a poroelastic-viscoelastic (PVE) model in Matlab to determine parameters of stiffness (E), viscoelastic ratio (E∞/E0), and intrinsic permeability (k) [10]. Following nanoindentation, all samples were stained for Masson’s Trichrome using standard protocols. A linear mixed-effects model (p < 0.05), with patient ID set as the random variable, was employed for statistical analysis in RStudio; multiple comparisons were assessed with a Tukey post-hoc test.

Results, Conclusions, and Discussions: Uterine fibroids exhibit increased collagen and decreased smooth muscle content compared to patient-matched myometrium (Fig. 1D). Uterine fibroids are surrounded by circumferentially aligned smooth muscle fibers in the adjacent myometrium, which is markedly distinct from the random fiber orientation displayed in the distant myometrium (Fig. 1D). Mechanically, seedling uterine fibroids are significantly stiffer and less permeable than adjacent and distant myometrium tissue, with no difference in viscoelastic ratio observed (Fig. 1E). A negative linear relationship between stiffness and permeability is revealed for fibroids; this correlation is absent for all myometrial tissues (Fig. 1G). Notable heterogeneity in material property values is observed within individual and across distinct fibroid specimens (Fig. 1E,F). The transition in stiffness values at the uterine fibroid-myometrium interface is variable by specimen and can be grouped into three main categories: (a) sharp, (b) gradual, or (c) absent (Fig. 1F).

The data presented in this study yield key insights into the mechanical and structural properties of seedling uterine fibroids on the microscale, with a focus on the fibroid-myometrial interface. These data suggest uterine fibroids have a unique biophysical fingerprint that differs from adjacent and distant myometrium tissues in terms of stiffness, permeability, and collagen content. Further, the distinct groupings of stiffness transitions at the fibroid-myometrial interface suggest the existence of multiple morphological phenotypes at the seedling fibroid stage. Overall, this study contributes to a foundational understanding of uterine fibroid pathophysiology, which is necessary for the development of diagnostic imaging tools and targeted therapeutic treatments. Future studies will focus on assessing the effect of certain patient and fibroid characteristics on the material properties of uterine fibroids including menstrual cycle stage, menopause status, exogenous hormone use, obstetric history, fibroid subtypes (i.e., submucosal, intramural, subserosal) and fibroid size.

Acknowledgements (Optional):

References: [1] Baird et al. Am J Obstet Gynecol. 2003. [2] Bulun, S. N Engl J Med. 2013. [3] Farquhar, C et al. Obstet Gynecol. 2002. [4] Stewart et al. Nat Rev Dis Primer. 2016. [5] Rogers et al. Am J Obstet Gynecol. 2008. [6] Norian et al. Matrix Biol. 2012. [7] Omari et al. J Biomech. 2015. [8] Jayes et al. Am J Obstet Gynecol. 2016. [9] Jayes et al. PLOS One. 2019. [10] Islam and Oyen. Exp. Mech. 2021.

Acknowledgements:
This work is supported financially by the Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health under award number 1R01HD091153, NSF Graduate Research Fellowship, and the Iris Fund. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health