Daniel F. Rychlik

A New Model for Inflammation-Induced Preterm Birth: The Role of Platelet-Activating Factor and Toll-Like Receptor-4

Preterm birth is a leading cause of neonatal morbidity and mortality. Despite a growing body of evidence correlating inflammation with preterm birth, the signal transduction pathways responsible for the emptying of the uterus in the setting of intrauterine inflammation has not been elucidated. We now report a unique, reproducible mouse model of localized intrauterine inflammation. This model results in 100% preterm delivery with no maternal mortality. Using our model, we also show that platelet-activating factor is a crucial mediator of both inflammation-induced preterm birth and fetal demise. Using C3H/HeJ mice, we demonstrate that toll-like receptor-4 (TLR-4) plays a role in lipopolysaccharide-induced preterm birth but not in inflammation-induced fetal death. Immunohistochemistry studies demonstrate the presence of the platelet-activating factor receptor in both endometrial glands and smooth muscle in uterine tissues. Molecular studies demonstrate the differential expression of platelet-activating factor receptor and TLR-4 in uterine and cervical tissue throughout gestation. Quantitative polymerase chain reaction revealed an up-regulation of TLR-4 in the fundal region of the uterus in response to intrauterine inflammation. The use of this model will increase our understanding of the significant clinical problem of inflammation-induced preterm birth and will elucidate signal transduction pathways involved in an inflammatory state.

Cloning and tissue expression of the tissue prothrombinase Fgl-2 in the Sprague-Dawley rat.

Objective: To sequence and characterize the expression of the prothrombinase Fgl-2 in the Sprague-Dawley rat. Methods: Reverse-transcriptase polymerase chain reaction was performed on RNA from spontaneoulsy cycling adult pregnant Sprague-Dawley rats by using specific Fgl-w primers. The resulting amplicon was also used to screen a rat spleen bacteriophage library and to probe a Northern blot of various tissues. The rat Fgl-2 amino acid sequence was compared with the known sequences in mouse and human. Results: Fgl-w specific amplicon bands were observed in the rat brain, kidney, liver, ovary, spleen, and gestational day 22 and postpartum uterus. The rat Fgl-2 cDNA and amino acid sequence were found to be homologous with those of human (86% and 74%, respectively) and mouse (91% and 87%, respectively). Northern blotting demonstrated two different-sized transcripts (1.3 and 3.4 kb), and expression was observed in the cevix, heart, liver, ovary, and nongestational and gestational day 22 myometrium. Conclusion: Thrombin is classically generated from the clevage of the proenzyme prothrombin by activated factors V and X. In tissues thrombin appears to be generated by a novel prothrombinase Fgl-2 (fibrinogen-like protein) whose activity is stimulated by proinflammatory mediators. Fgl-2 provides the mechanistic coupling between proinflammatory cytokines and generation of active thrombin independent of the coagulation cascade. Our studies confirmed the expression of Fgl-2 mRNA in several rat tissues, including the pregnant uterus, where it could play a key role in the initiation of parturition especially in response to local or systemic infection.

Thoracic Endometriosis Syndrome Resembling Pulmonary Embolism

A 32-year-old infertility patient with a previous diagnosis of stage IV endometriosis experienced shortness of breath and chest pain. She was diagnosed with a pulmonary embolism by spiral volumetric computed tomography (SVCT) and anticoagulated during hospitalization, although no history of thrombosis was ever identified. She continued to have intermittent symptoms of chest pain, back pain, and shortness of breath for the next 1.5 months. Repeat SVCT revealed a large, right-sided pleural effusion with associated consolidation but no evidence of pulmonary embolism. To obtain a definitive diagnosis, a thoracoscopic pleural biopsy was performed and showed thoracic endometriosis involving the pleura. The patient desired to retain her fertility and opted for treatment with depot medroxyprogesterone. She has been asymptomatic for 2 years with this treatment. This case illustrates the importance of recognizing thoracic endometriosis syndrome and the difficulty diagnosing this condition considering its nonspecific features.

Constitutive Androstane Receptor Expression in the Rat Cervix During Gestation

Objective: To investigate expression of the constitutive androstane receptor (CAR) in the pregnant rat cervix. Methods: Rat uterine tissue was obtained on gestational days 12, 16, 20, 21, and 22 (the day of parturition), and postpartum day 1. In addition, liver, lung, kidney, heart, and skeletal muscle tissue were obtained. Expression of the two known CAR isoforms was evaluated using reverse transcriptase-polymerase chain reaction. Results: These studies confirmed CAR expression in the liver; however, CAR was not demonstrated in the myometrium or cervical tissue. Conclusions: The currently described CAR1 and CAR2 isoforms are not expressed in rat uterine tissue; therefore, they do not appear to participate in parturition in the pregnant rat.