Mechanical and Biochemical Effects of Progesterone on Engineered Cervical Tissue

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 <i>in vitro</i>. 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^-8 M estradiol versus 10^-8 M estradiol +10^-6 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.