Intra-amniotic infection is strongly associated with adverse pregnancy and neonatal outcomes. Most intra-amniotic infections are due to Ureaplasma species; however, the pathogenic potency of these genital mycoplasmas to induce preterm birth is still controversial. Here, we first laid out a taxonomic characterization of Ureaplasma isolates from women with intra-amniotic infection, which revealed that Ureaplasma parvum is the most common bacterium found in this clinical condition. Next, using animal models, we provided a causal link between intra-amniotic inoculation with Ureaplasma species and preterm birth. Importantly, the intra-amniotic inoculation of Ureaplasma species induced high rates of mortality in both preterm and term neonates. The in vivo potency of U. parvum to induce preterm birth was not associated with known virulence factors. However, term-derived and preterm-derived U. parvum isolates were capable of inducing an intra-amniotic inflammatory response. Both U. parvum isolates invaded several fetal tissues, primarily the fetal lung, and caused fetal inflammatory response syndrome. This bacterium was also detected in the placenta, reproductive tissues, and most severely in the fetal membranes, inducing a local inflammatory response that was replicated in an in vitro model. Importantly, treatment with clarithromycin, a recently recommended yet not widely utilized antibiotic, prevented the adverse pregnancy and neonatal outcomes induced by U. parvum. These findings shed light on the maternal-fetal immunobiology of intra-amniotic infection. IMPORTANCE Preterm birth is the leading cause of neonatal morbidity and mortality worldwide. Multiple etiologies are associated with preterm birth; however, 25% of preterm infants are born to a mother with intra-amniotic infection, most commonly due to invasion of the amniotic cavity by Ureaplasma species. Much research has focused on establishing a link between Ureaplasma species and adverse pregnancy/neonatal outcomes; however, little is known about the taxonomy of and host response against Ureaplasma species. Here, we applied a multifaceted approach, including human samples, in vivo models, and in vitro manipulations, to study the maternal-fetal immunobiology of Ureaplasma infection during pregnancy. Furthermore, we investigated the use of clarithromycin as a treatment for this infection. Our research provides translational knowledge that bolsters scientific understanding of Ureaplasma species as a cause of adverse pregnancy/neonatal outcomes and gives strong evidence for the use of clarithromycin as the recommended treatment for women intra-amniotically infected with Ureaplasma species.

Successful pregnancy requires a tightly-regulated equilibrium of immune cell interactions at the maternal-fetal interface (i.e., the decidual tissues), which plays a central role in the inflammatory process of labor. Most of the innate immune cells in this compartment have been well characterized; however, adaptive immune cells are still under investigation. Herein, we performed immunophenotyping of the decidua basalis and decidua parietalis to determine whether exhausted and senescent T cells are present at the maternal-fetal interface and whether the presence of pathological (i.e., preterm) or physiological (i.e., term) labor and/or placental inflammation alter such adaptive immune cells. In addition, decidual exhausted T cells were sorted to test their functional status. We found that (1) exhausted and senescent T cells were present at the maternal-fetal interface and predominantly expressed an effector memory phenotype, (2) exhausted CD4+ T cells increased in the decidua parietalis as gestational age progressed, (3) exhausted CD4+ and CD8+ T cells decreased in the decidua basalis of women who underwent labor at term compared to those without labor, (4) exhausted CD4+ T cells declined with the presence of placental inflammation in the decidua basalis of women with preterm labor, (5) exhausted CD8+ T cells decreased with the presence of placental inflammation in the decidua basalis of women who underwent labor at term, (6) both senescent CD4+ and CD8+ T cells declined with the presence of placental inflammation in the decidua basalis of women who underwent preterm labor, and (7) decidual exhausted T cells produced IFNγ and TNFα upon in vitro stimulation. Collectively, these findings indicate that exhausted and senescent T cells are present at the human maternal-fetal interface and undergo alterations in a subset of women either with labor at term or preterm labor and placental inflammation. Importantly, decidual T cell function can be restored upon stimulation.

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Problem: Preterm birth is commonly preceded by preterm labor, a syndrome that is causally linked to both intra-amniotic infection and intra-amniotic inflammation.However, the stereotypical cellular immune responses in these two clinical conditions are poorly understood.Method of study: Amniotic fluid samples (n = 26) were collected from women diagnosed with preterm labor and intra-amniotic infection (amniotic fluid IL-6 concentrations ≥2.6 ng/mL and culturable microorganisms, n = 10) or intra-amniotic inflammation (amniotic fluid IL-6 concentrations ≥2.6 ng/mL without culturable microorganisms, n = 16). Flow cytometry was performed to evaluate the phenotype and number of amniotic fluid leukocytes. Amniotic fluid concentrations of classical proinflammatory cytokines, type 1 and type 2 cytokines, and T-cell chemokines were determined using immunoassays.Results: Women with spontaneous preterm labor and intra-amniotic infection had (a) a greater number of total leukocytes, including neutrophils and monocytes/macrophages, in amniotic fluid; (b) a higher number of total T cells and CD4 + T cells, but not CD8 + T cells or B cells, in amniotic fluid; and (c) increased amniotic fluid concentrations of IL-6, IL-1β, and IL-10, compared to those with intra-amniotic inflammation. However, no differences in amniotic fluid concentrations of T-cell cytokines and chemokines were observed between these two clinical conditions. K E Y W O R D S chorioamnionitis, fetal inflammatory response syndrome, funisitis, microbial invasion of the amniotic cavity, placental inflammation, pregnancy | 3 of 15 GOMEZ-LOPEZ et al. | Placental histopathological examinationPlacentas were examined histologically by perinatal pathologists blinded to clinical diagnoses and obstetrical outcomes according to standardized Perinatology Research Branch protocols. 75,76 Briefly, three to nine sections of the placenta were examined, and at least one full-thickness section was taken from the center of the placenta; others were taken randomly from the placental disk. Acute inflammatory lesions of the placenta (maternal inflammatory response and fetal inflammatory response) were diagnosed according to established criteria, including staging and grading. 75,77 The proportions of patients whose placentas presented acute maternal and/or fetal inflammatory responses are displayed in Table 1. | Amniotic fluid sample collectionAmniotic fluid samples were obtained by transabdominal amniocentesis under antiseptic conditions and monitored by ultrasound in order to detect intra-amniotic inflammation and/or infection in patients with preterm labor. Samples of amniotic fluid were transported to the laboratory in a sterile, capped syringe and immunophenotyping was performed immediately. The rest of the sample was centrifuged at 1300 g for 10 minutes at 4°C, and the supernatant was stored at −80°C until use. Also, an aliquot of amniotic fluid was transported to the clinical laboratory for culture of aerobic/anaerobic bacteria and genital mycoplasmas. The clinical tests also inc...

Background Monocytes, after neutrophils, are the most abundant white blood cells found in the amniotic cavity of women with intra-amniotic inflammation/infection. However, the origin of such cells has not been fully investigated. Herein, we determined (1) the origin of amniotic fluid monocytes/macrophages from women with intra-amniotic inflammation/infection, (2) the relationship between the origin of amniotic fluid monocytes/macrophages and preterm or term delivery and (3) the localization of monocytes/macrophages in the placental tissues. Methods Amniotic fluid samples (n = 16) were collected from women with suspected intra-amniotic inflammation or infection. Amniotic fluid monocytes/macrophages were purified by fluorescence-activated cell sorting, and DNA fingerprinting was performed. Blinded placental histopathological evaluations were conducted. Immunohistochemistry was performed to detect CD14+ monocytes/macrophages in the placental tissues. Results DNA fingerprinting revealed that (1) 56.25% (9/16) of amniotic fluid samples had mostly fetal monocytes/macrophages, (2) 37.5% (6/16) had predominantly maternal monocytes/macrophages and (3) one sample (6.25% [1/16]) had a mixture of fetal and maternal monocytes/macrophages. (4) Most samples with predominantly fetal monocytes/macrophages were from women who delivered early preterm neonates (77.8% [7/9]), whereas all samples with mostly maternal monocytes/macrophages or a mixture of both were from women who delivered term or late preterm neonates (100% [7/7]). (5) Most of the women included in this study presented acute maternal and fetal inflammatory responses in the placenta (85.7% [12/14]). (6) Women who had mostly fetal monocytes/macrophages in amniotic fluid had abundant CD14+ cells in the umbilical cord and chorionic plate, whereas women with mostly maternal amniotic fluid monocytes/macrophages had abundant CD14+ cells in the chorioamniotic membranes. Conclusion Amniotic fluid monocytes/macrophages can be of either fetal or maternal origin, or a mixture of both, in women with intra-amniotic inflammation or infection. These immune cells could be derived from the fetal and maternal vasculature of the placenta.

Abstract Sterile intra-amniotic inflammation is a clinical condition frequently observed in women with preterm labor and birth, the leading cause of neonatal morbidity and mortality worldwide. Growing evidence suggests that alarmins found in amniotic fluid, such as interleukin (IL)-1α, are central initiators of sterile intra-amniotic inflammation. However, no causal link between elevated intra-amniotic concentrations of IL-1α and preterm birth exists. Herein, using an animal model of ultrasound-guided intra-amniotic injection of IL-1α, we show that elevated concentrations of IL-1α cause preterm birth and neonatal mortality. Additionally, using immunoblotting techniques and a specific immunoassay, we report that the intra-amniotic administration of IL-1α induces activation of the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in the fetal membranes, but not in the decidua, as evidenced by a concomitant increase in the protein levels of NLRP3, active caspase-1, and IL-1β. Lastly, using Nlrp3-/- mice, we demonstrate that the deficiency of this inflammasome sensor molecule reduces the rates of preterm birth and neonatal mortality caused by the intra-amniotic injection of IL-1α. Collectively, these results demonstrate a causal link between elevated IL-1α concentrations in the amniotic cavity and preterm birth as well as adverse neonatal outcomes, a pathological process that is mediated by the NLRP3 inflammasome. These findings shed light on the mechanisms underlying sterile intra-amniotic inflammation and provide further evidence that this clinical condition can potentially be treated by targeting the NLRP3 inflammasome.

Objective: Preterm birth is the leading cause of neonatal morbidity and mortality worldwide. Some preterm births are associated with clinical chorioamnionitis; yet, this condition has been poorly investigated. Herein, we characterized the amniotic fluid cellular immune responses in women with preterm clinical chorioamnionitis.Methods and subjects: Amniotic fluid samples were obtained from women with preterm clinical chorioamnionitis and a positive or negative microbiological culture (n=17). The cellular composition of amniotic fluid was evaluated using fluorescence microscopy, scanning

Background Preterm birth is the leading cause of perinatal morbidity and mortality. Preterm prelabor rupture of membranes (pPROM) occurs in 30% of preterm births; thus, this complication is a major contributor to maternal and neonatal morbidity. However, the cellular immune responses in amniotic fluid of women with pPROM have not been investigated. Methods Amniotic fluid samples were obtained from women with pPROM and a positive (n = 7) or negative (n = 10) microbiological culture. Flow cytometry was performed to evaluate the phenotype and number of amniotic fluid leukocytes. The correlation between amniotic fluid immune cells and an interleukin-6 (IL-6) concentration or a white blood cell (WBC) count in amniotic fluid was calculated. Results Women with pPROM and a positive amniotic fluid culture had (1) a greater number of total leukocytes in amniotic fluid, including neutrophils and monocytes/macrophages and (2) an increased number of total T cells in amniotic fluid, namely CD4+ T cells and CD8+ T cells, but not B cells. The numbers of neutrophils and monocytes/macrophages were positively correlated with IL-6 concentrations and WBC counts in amniotic fluid of women with pPROM. Conclusion Women with pPROM and a positive amniotic fluid culture exhibit a more severe cellular immune response than those with a negative culture, which is associated with well-known markers of intra-amniotic inflammation.