Proteomic analysis of amniotic fluid shows the presence of biomarkers characteristic of intrauterine inflammation. We sought to validate prospectively the clinical utility of one such proteomic profile, the Mass Restricted (MR) score.
Methods and Findings
We enrolled 169 consecutive women with singleton pregnancies admitted with preterm labor or preterm premature rupture of membranes. All women had a clinically indicated amniocentesis to rule out intra-amniotic infection. A proteomic fingerprint (MR score) was generated from fresh samples of amniotic fluid using surface-enhanced laser desorption ionization (SELDI) mass spectrometry. Presence or absence of the biomarkers of the MR score was interpreted in relationship to the amniocentesis-to-delivery interval, placental inflammation, and early-onset neonatal sepsis for all neonates admitted to the Newborn Special Care Unit (n = 104). Women with “severe” amniotic fluid inflammation (MR score of 3 or 4) had shorter amniocentesis-to-delivery intervals than women with “no” (MR score of 0) inflammation or even “minimal” (MR score of 1 or 2) inflammation (median [range] MR 3–4: 0.4 d [0.0–49.6 d] versus MR 1–2: 3.8 d [0.0–151.2 d] versus MR 0: 17.0 d [0.1–94.3 d], p < 0.001). Nonetheless, a “minimal” degree of inflammation was also associated with preterm birth regardless of membrane status. There was a significant association between the MR score and severity of histological chorioamnionitis (r = 0.599, p < 0.001). Furthermore, neonatal hematological indices and early-onset sepsis significantly correlated with the MR score even after adjusting for gestational age at birth (OR for MR 3–4: 3.3 [95% CI, 1.1 to 9.2], p = 0.03). When compared with other laboratory tests routinely used to diagnose amniotic fluid inflammation and infection, the MR score had the highest accuracy to detect inflammation (white blood cell count > 100 cells/mm3), whereas the combination of Gram stain and MR score was best for rapid prediction of intra-amniotic infection (positive amniotic fluid culture).
High MR scores are associated with preterm delivery, histological chorioamnionitis, and early-onset neonatal sepsis. In this study, proteomic analysis of amniotic fluid was shown to be the most accurate test for diagnosis of intra-amniotic inflammation, whereas addition of the MR score to the Gram stain provides the best combination of tests to rapidly predict infection.
Proteomic analysis of amniotic fluid in addition to a Gram stain provides the best combination of tests to rapidly predict intrauterine infection.
A preterm delivery, or premature birth, is normally defined as one that occurs before 37 weeks after the last menstrual cycle (an average pregnancy lasts around 40 weeks). Premature birth is fairly common, with around 12% of births in the US fitting this definition. However, it has serious consequences, being responsible for around 70% of infant deaths and other adverse outcomes for the baby. It is not clear in all cases what directly causes premature birth or how to identify cases in which mother and child are at greater risk of serious outcomes. Evidence from case-control and other studies strongly suggests that infections of the uterus, placenta, or genital tract are associated with, and are likely to directly cause, premature deliveries. Such infections, even if they are “subclinical” (that is, they do not directly cause signs or symptoms that the doctor or patient would notice) cause inflammation in the affected tissues. Hence, it's possible that particular proteins or other molecules could provide a “signature” that would allow the inflammation to be picked up at an early stage.
Why Was This Study Done?
If inflammation could be picked up early, this might help identify mothers at risk of having a preterm delivery, and even to pinpoint cases of very severe inflammation where the baby is more at risk of poor outcomes. The researchers involved in this study had already done previous work looking at protein profiles in the amniotic fluid (the liquid directly surrounding the developing fetus). They identified a set of four protein “markers” that were closely associated with inflammation in the amniotic fluid, and developed a score based on those proteins, which they termed the “Mass Restricted” (MR) score. The researchers showed that this score could accurately identify women at risk of preterm delivery. However, before using the protein marker score in clinical practice it is very important to really be sure it is a reliable diagnostic test for preterm birth and adverse outcomes resulting from preterm birth. Therefore the researchers wanted to find out whether MR scores were associated with the outcome of pregnancy; the presence of infection in the placenta, as detected through microscopic analysis of tissue; and sepsis (severe infection) in the newborn baby.
What Did the Researchers Do and Find?
The study was based on findings from pregnant women presenting at the Yale-New Haven Hospital with symptoms of premature labor, who were all followed up to the point of delivery of the baby. In all cases the decisions about how to manage the pregnancy (for example, whether to deliver the baby or attempt to delay birth) were made by the woman and her physician, not by any procedures laid out in the research study. A total of 169 women were recruited into the study and had a sample of amniotic fluid taken as part of their routine clinical management. The researchers then analyzed this fluid to calculate the protein MR score, to look for evidence of bacterial infection, and also carried out standard laboratory tests. After childbirth the placenta was examined under the microscope to look for any evidence of inflammation. Finally, all babies were checked for any evidence of sepsis. The researchers found that, in line with findings from their previous studies, women with a higher MR score gave birth sooner. There also seemed to be a close agreement between the MR score and evidence of inflammation in the placenta, once it was analyzed under the microscope after birth. Furthermore, mothers with a high MR score were more likely to give birth to babies with suspected or confirmed sepsis. The researchers then compared the usefulness of the MR score against other potential tests for inflammation. Of all the tests compared, the MR score seemed to be the most accurate in predicting inflammation.
What Do These Findings Mean?
This study showed that the MR score was closely associated with a number of different indicators of poor outcome in preterm birth. These outcomes included sooner deliveries, sepsis in the baby, and inflammation in the placenta. In future, the MR score may provide a useful test for recognizing women at risk of preterm delivery and babies at risk of poor outcome. However, further evaluation of the test will still need to be done before it could become a standard procedure in the clinic.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040018.
Information from the US National Institutes of Health on premature babies
The March of Dimes is a US charity that funds research into prematurity
Information from Wikipedia about proteomics the area of research used to develop the protein score examined here (note: Wikipedia is an online encyclopedia that anyone can edit)