The Role Of Subclinical Infection And Cytokines In Preterm Parturition

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

I. Understanding the mechanisms of human term and preterm parturition through transcriptomics A. A single-cell atlas of the myometrium in human parturition (17, 29) Few biological processes as central to the survival of viviparous species are so incompletely understood as parturition. Yet, there is an inadequate understanding of the physiology of normal labor and the pathophysiology of labor disorders: preterm and term. The Branch has utilized bulk transcriptomics to understand the components of the common pathway of parturition, which include myometrial contractility, cervical remodeling, and membrane/decidual activation, and has characterized differentially expressed genes and biological processes enriched during parturition. We have now leveraged single-cell technology to assemble a human cell atlas of parturition and have analyzed the placenta and chorioamniotic membranes. However, the engine of labor is the myometrium, which is inaccessible for study. Therefore, one of our goals has been to determine whether it is possible to monitor cellular activity in the myometrium non-invasively through maternal blood. We performed RNA single-cell sequencing and generated the first single-cell atlas of the human myometrium during labor as well as the first map of cell type-specific transcriptomic activity modulation. This information provides the foundation to study labor disorders. Integrating scRNA-seq information with transcriptomic data derived from bulk analyses of the myometrium, we characterized the contributions of smooth muscle cells and inflammation during labor. Importantly, we showed that myometrium derived single-cell signatures could be detected and quantitated in maternal blood. This result provides evidence that it is possible to monitor myometrial biology non-invasively by interrogating maternal blood. Indeed, we have developed a transcriptomic signature of labor in maternal blood in term and preterm labor. II. The proteome of human pregnancy and preterm delivery A. The amniotic fluid proteome in normal pregnancy and impending preterm delivery (2, 20) We have studied the amniotic fluid proteome to establish a systems biology approach for pregnancy complications. We first characterized the amniotic fluid proteome and found that about 25% (320/1310) of proteins changed significantly in abundance with gestational age. Intersecting gestational age-modulated proteins and their corresponding mRNAs previously reported in the maternal blood identified neutrophil-related protein/mRNA pairs modulated in the same direction. Our observations have implications for the discovery of biomarkers to diagnose obstetrical and fetal disorders. A sonographic short cervix, the most powerful predictor of spontaneous preterm birth, is an indication for treatment with vaginal progesterone to prevent preterm birth. However, only a fraction of women responds to progesterone. Therefore, markers are required to determine which patients with a sonographic short cervix will progress to imminent delivery. We conducted a study to identify proteomic signatures of impending preterm delivery in patients with an asymptomatic sonographic short cervix (<25 mm) at 16 to 32 weeks of gestation. A combination of four amniotic fluid proteins (CXCL8, SNAP25, PTPN11, and MMP8) had a sensitivity of 79% with a 10% false-positive rate for the prediction of delivery within two weeks. This was a substantial improvement over the prediction derived from cervical length alone. We have already developed rapid point-of-care tests for the determination of CXCL8 and MMP-8 in amniotic fluid. These observations have clinical implications given the recent evidence generated by our group that intra-amniotic inflammation can be eradicated leading to prolongation of pregnancy. In addition, the diagnosis of silent intra-amniotic inflammation has implications for the management of the patient presenting with an asymptomatic short cervix. III. Novel molecular and cellular treatments to prevent premature labor and fetal injury A. Treatment of sterile intra-amniotic inflammation with clarithromycin prevents preterm delivery and reduces neonatal morbidity through modulation of inflammatory responses in maternal and fetal tissues (10) Sterile intra-amniotic inflammation has emerged as the most frequent identifiable etiology of spontaneous preterm labor with intact membranes and cervical insufficiency. Recent clinical studies from the Branch have demonstrated that successful treatment of sterile intra-amniotic inflammation can be achieved with clarithromycin, an antimicrobial agent with strong anti-inflammatory properties. We have conducted a number of studies to determine the mechanisms by which clarithromycin prolongs gestation and improves neonatal outcomes. Using a previously established animal model of sterile intra-amniotic inflammation generated in our laboratory, we found that clarithromycin prolonged gestation, reduced the rate of preterm birth, and improved neonatal survival. Clarithromycin also exhibited potent anti-inflammatory effects in the placental and fetal tissues, which may contribute to the improved neonatal outcomes. These findings provide mechanistic evidence that clarithromycin can be administered to prevent preterm birth and to improve neonatal survival in the context of sterile intra-amniotic inflammation. B. Maternal macrophages have a role in the maintenance of pregnancy and the potential use of M2-polarized macrophages as cellular therapy to protect against inflammation-induced premature labor and fetal injury (16) Macrophages participate in the mechanisms of preterm and term labor by amplifying inflammation; however, there is a considerable body of evidence that a subset of macrophagestypically referred to as M2 macrophageshave powerful anti-inflammatory effects. We undertook a series of experiments to study the role of macrophages in pregnancy and observed that 1) women in labor had a decreased number of M2-like macrophages at the maternal-fetal interface; 2) depletion of maternal CD11b+ myeloid cells led to preterm labor and adverse neonatal outcomes, and this was ameliorated by replacement of wild-type macrophages; 3) adoptive transfer of M2-polarized macrophages in vitro reduced the incidence of preterm birth and improved neonatal survival in a model of intra-amniotic inflammation; and 4) M2-polarized macrophages downregulated the inflammatory response in the fetal brain and lungs (major target organs in the fetal inflammatory response syndrome). These results demonstrate a hitherto unappreciated homeostatic role for macrophages in the physiology of pregnancy and a therapeutic role for M2-polarized macrophages for the treatment of premature labor and of the fetal inflammatory response syndrome. IV. Refining the use of progesterone for the prevention of preterm birth (5, 6, 31) Our previous studies have shown that vaginal progesterone reduces the rate of preterm birth in women with a sonographic short cervix in the midtrimester and decreases neonatal morbidity. Some professional organizations have proposed that vaginal progesterone should also be used in patients who have a history of preterm birth regardless of cervical length. Therefore, we conducted two meta-analyses and showed that vaginal progesterone is not efficacious in patients with a prior history of preterm birth or in those without a short cervixthis has immediate implications for clinical practice.