Mechanical and Biochemical Effects of Progesterone on Engineered Cervical Tissue

Abstract

Preterm birth is a leading cause of morbidity and mortality in newborns. Babies born prematurely are at increased risk of lifelong health problems, including neurodevelopmental abnormalities. Cervical shortening precedes preterm birth in many women. Cervical shortening is caused, in part, by excessive softening of the extracellular matrix (ECM) of the cervical stroma. In clinical obstetrics, cervical shortening prompts treatment with supplemental progesterone to prevent preterm birth. However, progesterone-mediated effects on the cervical ECM are not well understood. This research sought to study progesterone-mediated remodeling of ECM produced by human cervical fibroblasts in vitro. A previously developed three-dimensional (3D) engineered model of the cervical ECM was used for experiments. Cervical fibroblasts were seeded on porous scaffolds and cultured in spinner flasks to promote ECM synthesis. Scaffolds were exposed to two conditions: 10 M estradiol versus 10 M estradiol +10 M progesterone for 4 weeks. To measure ECM strength, two scaffolds were mounted end-to-end on a wire and cultured such that ECM filled the gap between the scaffolds. The force required to pull the scaffolds apart was measured. Collagen content and collagen crosslinks were measured with ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry. Whole-transcriptome RNA sequencing (RNA-seq) was used to quantify gene expression between the two experimental conditions. Zymography was used to study the quantity and activity of matrix metalloproteinase-2 (MMP2) in the scaffolds. The study found that exposure to progesterone increased tissue softness of the engineered ECM over 28 days. Increased tissue softness correlated with decreased collagen content. With RNA-seq, progesterone exposure resulted in gene expression changes consistent with known progesterone effects. Pathway analysis of the RNA-seq data suggested MMPs were significantly dysregulated in progesterone-exposed engineered ECM. Increased expression of active MMP2 was confirmed in the progesterone-exposed engineered ECM. In summary, progesterone increased the softness of the ECM, which was correlated with decreased collagen production and altered histology. These results are important for deciphering the role of progesterone in preventing preterm birth.

Mechanics of cervical remodelling: insights from rodent models of pregnancy

“…stretch or stress), identifying particular homeostatic state(s) cells work to maintain, and understanding how pregnancyinduced changes in biological and mechanical cues combine to affect intracellular signalling and alter gene expression and/or protein production. In vitro studies using three-dimensional cervical stromal cell culture systems [94] will provide a useful platform to study how mechanics, along with cellular signals, drive cervicalcell specific functions to achieve the dynamic and multi-factorial changes required in cervical remodelling.…”

Roadmap on biomaterials for women’s health

“…To address the limitations of current models of cervical softening during pregnancy, engineered models of cervical tissue are being investigated [133]. During pregnancy, it is known that cervical tissue is exposed to multiple factors that could decrease cervical stiffness including changes in hormone concentrations, inflammatory conditions, and mechanical loading.…”

“…After 4 weeks, the seeded scaffolds produced an ECM with biochemical components and histological appearance similar to native cervical stroma. Using the engineered cervix model, the influence of steroid hormones on ECM properties was studied [133]. The engineered cervix model suggested estradiol promoted ECM growth and progesterone had a softening effect.…”

Biomaterial strategies to replicate gynecological tissue

“…2D). 110 3D in vitro models of this tissue have been used to evaluate cell-ECM interactions. Mechanical and biochemical effects of progesterone on cervical tissue were…”

“…By measuring collagen content and crosslinking in the scaffolds after stimulation with progesterone, they demonstrated that fibroblasts changed their morphology and decreased their production of collagen, leading to a decrease in cervical tissue stiffness. 110 An in vitro model commonly used to study skin has also been employed to study cervical cancer: organotypic raft cultures. Organotypic raft cultures are multilayered models of collagen, fibroblasts, and keratinocytes that are maintained at an air-liquid interface.…”