Huiling Ji

Prostaglandin E2-regulated cervical ripening: analysis of proteoglycan expression in the rat cervix.

OBJECTIVE Prostaglandins reduce cervical resistance by reorganizing collagen fibrils. Proteoglycans are involved in collagen fibril organization and structure. We evaluated the changes in proteoglycan composition induced by prostaglandin E(2) (PGE(2)). STUDY DESIGN Prostaglandins were administered intravaginally to induce cervical ripening in timed pregnant Sprague-Dawley rats. Changes in proteoglycan messenger ribonucleic acid (mRNA) expression were measured using reverse transcription (RT-PCR) for core protein. Fluorophore assisted carbohydrate gel electrophoresis (FACE) was used to evaluate proteoglycan glycosaminoglycan composition along with size exclusion high-performance liquid chromatography (HPLC). RESULTS No change in core protein mRNA expression was detected after PGE(2) treatment. Total glycosaminoglycan (GAG) decreased more than 20% after PGE(2) (P = .02). FACE demonstrated a shift in disaccharide subunit composition after PGE(2), with a decrease in 4-sulfated disaccharides (P = .02). HPLC confirmed a decrease in total GAG (P = .04). CONCLUSION Although there was no change in core protein mRNA expression, alterations in GAG composition was detected after PGE(2). The decrease in sulfated GAG could decrease electrostatic interactions that would weaken interfibrillar interactions. These findings would be consistent with a decline in cervical resistance.

Androgen-regulated cervical ripening: a structural, biomechanical, and molecular analysis

OBJECTIVE Androgens regulate biomechanical responses in load-bearing tissues. Evidence suggests that androgens may play a role in the cervix. We hypothesized that androgens directly regulate cervical remodeling by altering both collagen structure and proteoglycan composition. STUDY DESIGN Cervical resistance was evaluated using the cervical creep method after the administration of intravaginal dihydrotestosterone or oral flutamide. Microstructural changes in collagen were evaluated by transmission electron microscopy and polarized light birefringence. Proteoglycan expression was evaluated by reverse transcription-polymerase chain reaction for the core proteins (decorin, biglycan, fibromodulin, aggrecan, versican) and fluorophore-assisted carbohydrate analysis. RESULTS Dihydrotestosterone decreased cervical resistance, whereas flutamide inhibited the decline in cervical resistance, compared with vehicle controls. Flutamide was associated with higher levels of organized collagen and increased aggrecan expression with a greater proportion of chondroitin/dermatan sulfate glycosaminoglycans. Flutamide inhibited the increase in hyaluronan. CONCLUSION Androgens appear to play a role in regulating cervical resistance by altering proteoglycan content. Structural analysis indicates that flutamide may alter collagen fibril organization and/or structure.

Progesterone Modulates Integrin α2 (ITGA2) and α11 (ITGA11) in the Pregnant Cervix

Objective: Fibrillar collagen in the cervical extracellular matrix (ECM) is the predominant component providing mechanical support. Cellular integrins contribute to structural integrity by cross-linking ECM components. We investigated the expression of collagen-binding integrins in the normal rat gestation and after treatment with mifepristone to determine whether integrin modulation is involved in changes in tissue resistance. Study design: Cervical tissue was harvested from nonpregnant and timed pregnant Sprague-Dawley rats. Normal gestational expression was evaluated in nonpregnant and timed pregnant tissue on days 12, 16, 18, 20, 21 and 22. Progesterone inhibition was induced with 3 mg mifepristone administered on day 15. Primary rat cervical stromal (RCS) cell cultures were generated from nonpregnant rats using tissue explants. The effects of progesterone environment on RCS cells were evaluated in the presence and absence of various inhibitors. Protein expression and signaling pathways were evaluated by Western blot. Results: Integrin α2 (ITGA2) expression increased over gestation, peaking at the end of gestation (analysis of variance [ANOVA] P < .01). Integrin α11 (ITGA11) expression increased through mid-gestation, peaking on day 18 and decreasing through day 22 (ANOVA P < .001). Progesterone increased the expression of ITGA11 and phosphorylated focal adhesion kinase ([pFAK] P < .002). Mifepristone blocked these effects in vitro. Mifepristone increased ITGA2 and phosphorylated extracellular signal-regulated kinases 1 and 2 (pERK1/2) in vivo and in vitro. Mifepristone-induced upregulation of ITGA2 was abrogated by inhibition of ERK1/2. Conclusion: Progesterone/progesterone withdrawal is involved in regulating the expression of collagen-binding integrins. These changes differ among the collagen-binding integrins. Mitogen-activated protein kinase (MAPK) signaling is involved in regulating some of these integrins.

Phospholipid Scramblase Isoform Expression in Pregnant Rat Uterus

Objective: Phospholipid scramblases (PLSCRs), a family of novel membrane proteins, facilitate the translocation of aminophospholipids from the inner to the exterior leaf of the cell membrane. Four isoforms of PLSCR (PLSCR1-4) have been reported in mouse and human. The studies described in this report sought to characterize the uterine expression of the PLSCR isoforms in the near-term pregnant rat. Methods: Uterine tissue was obtained from timed-pregnant Sprague-Dawley rats. Total RNA was isolated, treated with DNase, and used in qualitative and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) studies utilizing PLCSR isoform PCR primers. A rat spleen cDNA bacteriophage library was used as a template for PCR-based sequencing to determine the cDNA and translated amino acid sequences for the PLSCR3 and PLSCR4 homologs expressed in rats. The 5′ and 3′ untranslated regions were obtained using 5′ and 3′ Rapid Amplification of cDNA Ends (RACE) techniques. Results: RT-PCR studies confirmed expression of PLSCR3 and PLSCR4 in the endometrial and myometrial layers of the pregnant rat uterus; in contrast, PLSCR1 and PLSCR2 were not found in uterine tissues. The cDNA sequence for the rat PLSCR3 homolog was found to be 1642 nucleotides, having 92% identity with mouse and 80% with human PLSCR3. The rat PLSCR4 homolog has a cDNA sequence of 1879 nucleotides, having an 89% identity with mouse and 72% identity with human PLSCR4 homologs. Conclusion: The intrauterine expression of PLSCR3 and PLSCR4 provides a dynamic mechanism by which aminophospholipid translocation can be regulated, thereby modulating the activity of various membrane proteins that are involved in inflammation and coagulation-related events.

Effects of labor on placental fatty acid β oxidation

Objective: To measure the effect labor exerts on fatty acid (FA) oxidation in term human placentas, and to compare enzymes expression and activity between placenta and liver. Methods: Placental samples were collected: (a) scheduled non-labored cesarean section and (b) normal vaginal delivery at or beyond 37 weeks. Long and medium-chain FA oxidation were measured using 3H-labeled FA, ATP concentration was measured via commercial kit. Activity and expression levels of 11 FA enzymes were measured and results compared to both human and mouse liver. Results: Placentas undergoing labor had significantly decreased palmitate oxidation and ATP levels. Octanoic acid oxidation was 10-fold higher than palmitic acid oxidation. No difference in expression or activity level was detected between the groups. Conclusion: Term human placentas express all the enzymes required to oxidize FA, at a rate 20-fold lower than liver. FA Oxidation is not likely an important placental energy source during labor. Further work is needed to determine the functionality of this pathway in placenta.

THE ROLE OF TRANSFORMING GROWTH FACTOR β IN CERVICAL REMODELING WITHIN THE RAT CERVIX

OBJECTIVE Transforming growth factor beta (TGFbeta) plays a central role in extracellular matrix remodeling. We hypothesized that TGFbeta signaling is involved in cervical remodeling. This study evaluated patterns within this signaling pathway. STUDY DESIGN The cervices of nonpregnant and timed pregnant rats were obtained. Messenger ribonucleic acid (mRNA) expression of TGFbeta1, TGFbeta receptor 1 (TbetaR1), TbetaR2, and TbetaR3 was evaluated. Four animals were euthanized for each time point. Western blotting was performed for protein expression. Phosphorylated mothers against decapentaplegic (Smad)-2 and -3 phosphorylation was assessed to evaluate TGFbeta activation. RESULTS TGFbeta1 mRNA increased through day 21 and declined on day 22 (analysis of variance, P = .001). TbetaR1 expression was unchanged. TbetaR2 and TbetaR3 mRNA expression was similar to TGFbeta1. TbetaR3 protein expression was similar to mRNA. Smad2 phosphorylation paralleled changes in TbetaR3. CONCLUSION Components of the TGFbeta signaling pathway increase during pregnancy along with Smad2 activation. The decline on day 22 correlates with a transition to the ripening phase supporting a role in cervical remodeling.

OP04.04: Development of an in vivo quantitative ultrasound methodology to scan the cervix

B. L. McFarlin1, M. L. Oelze2, T. A. Bigelow3, E. K. Chien4, H. Ji5, W. D. O’Brien, Jr. 2 1Maternal Child Nursing, University of Illinois at Chicago, Chicago, United States, 2Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, Urbana, United States, 3Department of Electrical Engineering, University of North Dakota, Grand Forks, United States, 4Obstetrics and Gynecology, Brown University/Women’s and Infants Hospital of Rhode Island, Providence, United States, 5Department of Obstetrics and Gynecology, Brown University/Women’s and Infants Hospital of Rhode Island, Providence, United States