Biochemical Pathways and Myometrial Cell Differentiation Leading to Nodule Formation Containing Collagen and Fibronectin.

Utilizing both primary myometrial cells and a myometrial cell line, we show here that myometrial cells undergo transition to a myofibroblast-like phenotype after a biological insult of 72 hours serum starvation and serum add-back (SB: 1% to 10% FBS). We also found that thrombospondin-1 was increased and that the transforming growth factor-beta (TGFB)-SMAD3/4 pathway was activated. This pathway is a key mediator of fibrosis and extracellular matrix (ECM) deposition. Applying the same insult supplemented with TGFB3 (1-10ng/ml) and ascorbic acid (100µg/ml) in the serum add-back treatment, we further demonstrated that cells migrated into nodules containing collagen and fibronectin. The number of cellnodules was inversely related to the percentage serum add-back. Using transmission electron microscopy we demonstrated myofibroblast-like cells and fibril-like structures in the extracellular spaces of the nodules. This study is the first direct evidence of induction of myofibroblast transdifferentiation in cultured myometrial cells which is related to the increase of thrombospondin-1 (THBS1) and the activation of TGFBSMAD 3 / 4 pathways. Combined, these observations provide biochemical and direct morphological evidence that fibrotic responses can occur in cultured myometrial cells. The findings are the first to demonstrate uterine healing mechanisms at a molecular level. Our data support the concept that fibrosis may be an initial event in formation of fibroid which exhibits signaling pathways and molecular features of fibrosis and grow by both cellular proliferation and altered extracellular matrix accumulation. Our data assists in further understanding of myometrium tissue remodeling during gestation and postpartum.

Hyperglycosylated human chorionic gonadotropin does not increase progesterone production by luteinized granulosa cells.

CONTEXT: Trophoblast-derived human chorionic gonadotropin (hCG) promotes corpus luteum progesterone (P4) production, and wide ranges of serum P4 levels are noted in various pregnancy outcomes, despite similar hCG concentrations. There are five unique biologically active hCG variants in human pregnancy urine, and previous studies of P4 production in response to hCG have used only preparations containing all isoforms. Understanding exactly which hCG variant is primarily responsible for stimulating corpus luteum steroidogenesis may have great clinical and diagnostic implications, including in the setting of ectopic pregnancy. OBJECTIVE: Our objective was to delineate the role of the standard and hyperglycosylated (H)-hCG isoforms in stimulating P4 production by luteinized granulosa cells. DESIGN AND SETTING: Cell culture, ELISA, and fluorometric-based protein assays were done at Duke University Medical Center. PATIENTS: Patients were anonymous oocyte donors. INTERVENTION: Cultured luteinized granulosa cells were treated with 0.25, 0.5, and 1.0 ng/ml total hCG, which contains all isoforms, purified standard hCG (37.1 kDa), and purified H-hCG (42.8 kDa). MAIN OUTCOME MEASURE: P4 produced per total cellular protein (nanograms per microgram) was measured via ELISA and fluorometric protein determination kits. RESULTS: Both total hCG (P = 0.0003) and purified standard hCG (P < 0.0001) stimulated a dose-dependent increase in P4 production. Purified H-hCG did not change the P4 produced per total cellular protein response (P value not significant). CONCLUSIONS: Standard hCG stimulated P4 production by cultured granulosa cells and likely supports corpus luteum function via interactions with the LH/hCG receptor. In contrast, H-hCG did not increase P4 production, which indicates a nonsteroidogenic role for this protein during early gestation.

Progesterone protects fetal chorion and maternal decidua cells from calcium-induced death.

OBJECTIVE: The purpose of this study was to determine whether progesterone exerts a protective effect in chorion and decidua cells when exposed to calcimycin. STUDY DESIGN: Fetal membrane samples were collected from term elective repeat cesarean deliveries and chorion and decidua cells that are separated and cultured. Cells were pretreated with progesterone and exposed to calcimycin. Cell viability was determined, and percent cell viability was calculated. RESULTS: Exposure to calcimycin resulted in a reduction of cell viability in both chorion and decidua cells in a dose-dependent fashion. In chorion and decidua cells, progesterone pretreatment followed by calcimycin increased cell viability compared with calcimycin treatment alone (chorion, 67%, vs controls, 24%; P < .001; decidua, 58%, vs controls, 35%; P < .001). The progesterone receptor antagonist, RTI 6413-49a, blocked the protective effect of progesterone in both chorion and decidua cells. CONCLUSION: These preliminary results suggest that progesterone may provide a protective effect in fetal membrane cells and that this effect may be mediated through the progesterone receptor.

Characterization of progesterone receptor isoform expression in fetal membranes.

OBJECTIVE: To quantify expression of progesterone receptor (PR) messenger RNA (mRNA) isoforms in fetal membranes, and to determine whether these levels change in culture. STUDY DESIGN: Placentas from women undergoing term cesarean delivery before labor were collected. Layers of amnion, chorion, and decidua were separated manually, enzymatically digested, and separated further with the use of a density gradient. RNA was extracted immediately and after culture for 48 hours, then analyzed by quantitative reverse transcription polymerase chain reaction for PR-A, PR-B, and beta-2 microglobulin mRNA expression. Separation of cell types was confirmed by immunohistochemistry. RESULTS: PR isoform expression was identified in fetal membranes, with levels highest in decidua and below the limits of detection in amnion. The ratio of PR-A/PR-B mRNA was not significantly different between cell layers. PR mRNA isoform levels did not differ significantly in fresh versus cultured cells. CONCLUSION: Quantitative reverse transcription polymerase chain reaction was used to quantitate expression of PR mRNA isoforms in cells of fetal membranes and to validate systems for further study of PR with respect to inflammation, infection, and preterm delivery.