FINITE DEFORMATIONS OF THE PREGNANT ENTIRE MURINE REPRODUCTIVE TRACT UNDER INFLATION

Introduction: Due to ethical considerations, the female reproductive tract’s unique ability to undergo significant remodeling and large deformations during pregnancy has been understudied in human subjects. Researchers rely on animal models such as rodents, ewes, swine, and primates to investigate the mechanical properties of the reproductive organs throughout pregnancy [1]. Ex vivo reproductive tissues from rodents have been used to quantify the biaxial mechanical properties of the isolated vagina and uterine body using inflation testing [2-5]. However, deformations in these studies were characterized only partially [4,5], providing an incomplete picture of the in vivo mechanical behavior of the organ. Rodents can be adopted as a model for pregnancy research as long as differences between mice and humans are well understood. Thus, there is a need for foundational knowledge of normal pregnancy in rodents. We have recently documented pregnancy-induced in-vivo remodeling of the reproductive organs in mice using advanced MRI technology by taking advantage of mice’s short gestation period, ease of animal husbandry, and low cost [2]. In this study, we continue filling the knowledge gap on pregnancy by performing ex vivo inflation testing of the entire murine reproductive tracts with non-contact full-field deformation measurements to better mimic in vivo loading conditions.

Materials and

Methods: Reproductive tracts were isolated from virgin (n=10) and pregnant (gestation day 16, n=2) CD1 mice and mounted onto a dispensing needle via the introitus. The ovaries, oviducts, and the tips of the uterine horns were occluded with methyl 2-cyanoacrylate before being tied at the ends of the uterine horn (embryos were removed when present). Specimens were speckled with an aerosol black paint for non-contact strain measurement. Testing was performed on a custom-built inflation setup described elsewhere [3,4]. Briefly, specimens were immersed in a phosphate-buffered saline (PBS) filled tank, connected to a pressure transducer (Omega Engineering Inc., Norwalk, CT) and syringe pump (NewEra Pump Sytstems, Inc., Farmigdale, NY) via plastic tubing. Each tract was preconditioned by PBS infusion to a pressure of 0 to 10 kPa at a flow rate of 2 ml/min [4]. Tissues were unloaded to 0 kPa and infused until rupture (flow rate of 0.47 ml/min ). Non-contact strain measurements were computed on the dorsal side of the specimen from images using a stereo camera setup (Basler Inc., Exton, PA) with a calibrated 3D digital image correlation system (Vic-3D 9, Correlated Solutions, Columbia, SC). The average Lagrangian principal strains were collected over small circular regions at the vagina, uterine body (connection of the uterine horns to the cervix and vaginal fornix), and uterine horns (Figure 1A). Statistical significance (p < 0.05) for the nonpregnant data was determined with a mixed effects model and an uncorrected Fisher’s LSD test for multiple comparisons (GraphPad Prism 10, Dotmatics, Boston, MA).

Results, Conclusions, and Discussions: The average pressure at rupture and average volume infused in the non-pregnant reproductive tract (n=10) was 77.24 ± 19.35 kPa and 0.687 ± 0.155 ml. Tissue rupture occurred within the uterine horn wall, away from the introitus, the clamped end. The strains were non-linearly correlated with the applied pressure. The maximum principal strain was significantly larger (p < 0.05) than the minimum strain in each region of the non-pregnant reproductive tract (Figure 1B). Differences in the magnitude of maximum and minimum principal strains in the vagina align with the relationship between the hoop and axial strains, respectively, of other previous inflation studies of the ex vivo rat [2,3] and mouse vagina [4]. The uterine horns and uterine body had a more variable pressure-strain relationship across specimens, likely due to their overall irregular geometry and variations in wall thickness. Significant differences were observed across regions of the uterine body and the vagina at low pressures. Our strain values are higher than those reported in published studies on murine reproductive tissues due to differences in testing modality and applied pressures [4,5]. We are conducting additional tests on entire reproductive tracts from mice during gestation to observe differences due to pregnancy-induced remodeling. Preliminary results suggest that the pregnant reproductive tracts are much more distensible than the non-pregnant reproductive tracts. This is the first study that reports the full-field deformations of the entire reproductive tract, without isolating each organ, in response to increasing intraluminal pressure up to rupture. This work establishes new experimental methods for comparing the finite deformations at different regions of the reproductive tract, providing foundational knowledge in evaluating mice as an animal model for pregnancy. By better understanding murine pregnancy, we can generate new hypotheses of human pregnancy in the future to design new studies that use human specimens.

Acknowledgements (Optional): This work was funded by the National Science Foundation under grant # 2053851.

References:
[1] Baah-Dwomoh, A, App Mech Rev, 68(6), 2016. [2] McGuire, JA, Inter Foc, 9(4), 2019. [3] Dubik, J, Integr Comp Biol, 62(3), 625-640, 2022. [4] Robison, KM, J Biomech, 139(10), 2017. [5] Conway CK, J Biomech, 94, 39-48, 2019