Mark Phillippe

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.

Diabetic nephropathy and perinatal outcome

We studied the effect of diabetic nephropathy on the course of pregnancy, perinatal outcome, and infant development and determined the influence of pregnancy on maternal hypertension and renal function. Maternal proteinuria usually increased during pregnancy (greater than 3 gm/24 hours in 69%), and hypertension was present by the third trimester in 73%. The degree of proteinuria correlated with diastolic pressure and creatinine clearance. After pregnancy, proteinuria declined in 65% of the mothers, hypertension was absent in 43.5%, and the expected rate of fall in creatinine clearance was not accelerated. Among 35 patients, abortion occurred spontaneously or was performed electively in 25.7%, and 71% of the remainder underwent delivery before 37 weeks. Birth weight was related to maternal blood pressure and creatinine clearance. Neonatal morbidity was common, but the perinatal survival rate was 89%. Infants seen at follow-up without congenital anomalies had normal development at 8 to 36 months of age. We concluded that perinatal outcome has significantly improved for diabetic women with nephropathy.

Prematurity among insulin-requiring diabetic gravid women.

From Jan. 1, 1983, through Dec. 31, 1987, 420 gravidas with insulin-requiring diabetes antedating pregnancy delivered on the Joslin Clinic service. Among them, 110 pregnancies (26.2% of the total) delivered before 37 completed weeks of gestation compared with a 9.7% incidence (906/9368) for the general population at the Brigham and Womens Hospital during calendar year 1985. Thirty-three percent of all premature deliveries were the result of the development of preeclampsia. The relative risk of prematurity for diabetic patients with any hypertensive complication was 2.0 (95% confidence interval, 1.40 to 2.87) compared with normotensive diabetic subjects. Compared with the general population, most of the excess risk of prematurity was confined to hypertensive diabetics and normotensive patients of more advanced White class. A history of having had a previous premature delivery, increasing duration of diabetes antedating pregnancy, and carrying a male fetus in the index pregnancy were significantly associated with premature delivery. Future efforts to reduce the incidence of prematurity among diabetic gravidas should be directed toward reducing the incidence of preeclampsia.

Catecholamines in human amniotic fluid

This study was undertaken to determine the amniotic fluid levels of epinephrine (E), norepinephrine (N), and dopamine (D) at different gestational ages in the human. Amniotic fluid obtained from 32 pregnant women undergoing amniocentesis for medical indications was analyzed with a radioenzyme assay for these catecholamines. The gestational ages ranged from 17 to 39 weeks. No patient was in active labor at the time of amniocentesis. The data obtained in this study demonstrated a rise of all three catecholamines at the end of the third trimester: N = 362.7 +/- 61.2 pg/ml (+/- SEM), E = 179.6 +/- 45.4 pg/ml (p < 0.05), and D = 2717.0 +/- 836.6 pg/ml (p < 0.01). The physiologic role for the increasing amounts of catecholamines, especially D, in amniotic fluid is known; however, these amines could be the stimulus for the intrauterine synthesis of prostaglandins as parturition approaches.

Maternal mortality rate associated with cesarean section: an appraisal.

MODERN OBSTETRIC CARE includes a more liberalized use of cesarean section in the interest of fetal and neonatal outcome. Antepartum and intrapartum monitoring has enabled us to identify those fetuses better served by abdominal delivery. In other instances, retrospective analysis has taught that abdominal delivery is in the best interest of the fetus and neonate in cases of premature breech, midpelvic arrest, or other dystocias. This liberalized approach, which increased the incidence of cesarean section, has elicited concern from colleagues within our discipline, from other disciplines, from government, and from the public itself regarding increased maternal mortality rates, morbidity, and health care costs. Often such concern is inappropriately alarming and based on meager lay as well as scientific reports. This has prompted us to review our experience with maternal deaths associated with cesarean section. All births occurring at the Boston Hospital for Women for an 1 l-year period between 1968 and 1978 inclusive were tabulated from the official hospital records (Table I). A careful review of the records of the Committee on Maternal Welfare of the Massachusetts Medical Society verified that the data from the Boston Hospital for Women were complete. During this 1 l-year period, there were 68,645 births and 10,231 cesarean sections, both primary and repeat. The maternal mortality rate for those with cesarean section was zero. The maternal mortality rate was 6/58,414 births among those delivered vaginally (see Table II) and 6/68,645 births among the entire group, for a maternal mortality rate of 0.72/10,000. Assuming maternal deaths during this interval were distributed as a Poisson, there was no significant difference in the mortality rate between the two groups. In 1977, Evrard and Gold’ reported on the incidence of maternal deaths in Rhode Island during the lo-year period from 1965 to 1975. That experience encompassed 162,656 total deliveries and 12,941 cesarean sections. In the entire experience there were 20 obstetrics-related deaths, nine of which were associated with cesarean section. This report as well as other studie? 3 has suggested that the maternal mortality rate associ-

Randomized clinical trial of perioperative cefoxitin in preventing maternal infection after primary cesarean section

To determine the efficacy of perioperative cefoxitin in preventing infections after primary cesarean section, a randomized placebo-controlled, double-blind clinical trial was performed. Among 266 participants, those who received three perioperative 2 gm doses of cefoxitin (138) had significantly fewer serious infections (19.5% vs. 4.3%), fewer urinary tract infections (10.7% vs. 4.4%), less standard febrile morbidity (9.4% vs. 3.6%), and fewer courses of antibiotics postoperatively (23.4% vs. 11.6%). There was no reduction in the length of hospitalization. Use of perioperative cefoxitin umbilical cord is clamped are safe and efficacious in preventing infection after primary cesarean section.

Modulation of monocyte chemotactic protein-1 expression during lipopolysaccharide-induced preterm delivery in the pregnant mouse.

Preterm delivery is often associated with increased cytokine and chemokine production. These studies characterize the expression of the chemokine monocyte chemotactic protein-1 (MCP-1) in mice during lipopolysaccharide (LPS)—induced preterm delivery. Uterine and other tissues were harvested from CD-1 mice on gestational day 15 after intrauterine LPS injection. Quantitative real-time reverse-transcriptase polymerase chain reactions determined MCP-1 and toll-like receptor 4 (TLR4) mRNA expression during the 24 hours after LPS. MCP-1 protein expression was determined using a cytokine/chemokine protein array, enzyme-linked immunosorbant assay, and immunohistochemistry. Intrauterine LPS injection caused preterm delivery in CD-1 mice between 12 and 24 hours. Expression of MCP-1 mRNA significantly increased at 2 and 6 hours, while TLR4 expression did not significantly change over 24 hours. The MCP-1 protein levels peaked by 2 to 6 hours in maternal serum, liver, lung, kidney, and uterus. Immunohistochemistry confirmed MCP-1 in the myometrium and endometrium. These studies provide evidence suggesting that MCP-1 potentially plays an important role during the proinflammatory immune response, leading to preterm labor in the mouse.

Resistance to Lipopolysaccharide-Induced Preterm Delivery Mediated by Regulatory T Cell Function in Mice

Abstract Intrauterine or intraperitoneal administration of lipopolysaccharide (LPS) into normal mice at midgestation induces preterm delivery (PTD) within 24 h through a mechanism dependent on Toll-like receptor signaling and expression of inflammatory cytokines. The exact participants in the cellular network involved in PTD are not known. Although the activities of innate immune cells are thought to be important, the extent to which this process depends on T and B cells has yet to be examined. Mice deficient in T and B cells due to genetic deficiency in the recombination activating gene 1 (Rag1−/−) were given LPS intraperitoneally on Day 15 of gestation and found to be susceptible to LPS-induced PTD. This was found to involve many of the inflammatory mediators reported as important in normal mice. Moreover, at a low dose (3 μg), pregnant Rag1−/− mice were found to be more susceptible to PTD than a cohort of normal mice on the same genetic background. This increased susceptibility was partially reversed by transfer, on Day 10 of gestation, of whole lymphocytes or purified CD4+ T cells. Transfer of purified CD4+ T cells to Rag1−/− mice resulted in a uterine draining node population of FOXP3+ cells, suggesting that these cells may contribute to resistance to LPS-induced PTD. Overall, the data suggest that, although T and B lymphocytes are not critical positive regulators of LPS-induced PTD, CD4+ T cells play a protective and regulatory role, and thus could be a target for preventive or therapeutic manipulation.

Cell-free Fetal DNA — A Trigger for Parturition

Despite the importance of timing in parturition, little is known about its triggers. Emerging evidence supports an influential role for the level of cell-free fetal DNA.

Intracellular Signaling and Phasic Myometrial Contractions

This article reviews recently reported observations regarding the intracellular signal transduction mechanisms involved in the generation of phasic contractions occurring in myometrial tissue. The presence of cell surface receptors for classic uterotonic agonists (including oxytocin, norepinephrine, vasopressin, acetylcholine, and prostaglandins [PGs]) has been well described; all are seven-membrane-spanning, G protein-coupled receptors. Occupancy of these receptors, coupled through members of the Gq and/or Gi families of heterotrimetric G proteins, results in stimulation of the phospholipase C-β (PCL-β) isoforms. Nonclassic uterotonic agonists, such as growth factors and cytokines, also activate the phosphatidylinositol (PI)-signaling pathway, in this case through tyrosine kinase receptor-mediated activation of the phospholipase C-γ (PCL-γ) isoforms. Several recent reports have demonstrated that activation of the PI-signaling pathway in uterine myocytes results in the development of cytosolic calcium oscillation-like phenomena. These cytosolic calcium oscillations appear to arise from receptive cycles of emptying and refill of the endoplasmic reticulum calcium stores along with the influx of extracellular calcium. Calcium release from the endoplasmic reticulum calcium stores appears to be mediated by the inositol trisphosphate-sensitive and the ryanodine-sensitive receptor/channels; isoforms for both of these receptor/channels have been shown to be expressed in myometrial tissue. In summary, receptor-mediated activation of the PI-signaling pathway and the generation of cytosolic calcium oscillations appear to produce intermitten calcium transients that result in the development and maintenance of phasic myometrial contractions.