Proteomics/diagnosis of chorioamnionitis and of relationships with the fetal exposome

Abstract

Proteomics, a relatively young science, originally emerged as a complement to genomics research. By definition, the goal of proteomics is to provide a snapshot of all the proteins within an organism, tissue or biological sample at a given moment. Proteomics has the ability to single out one or more proteins (biomarkers) that change consistently in affected subjects as compared to those disease-free. From a proteomics perspective, chorioamnionitis poses both challenges and opportunities. Challenges relate to the dynamic course of the inflammatory process, and compartmentalization of the gestational sac in relation to the maternal compartment. An inability to evaluate the amniotic fluid non-invasively and repeatedly for meaningful changes in its proteome, and lack of a true gold standard for diagnosis of inflammation and/or infection, represent additional challenges. On the other hand, the unbiased and holistic nature of proteomics offers a real opportunity to improve the current diagnostic and prognostic algorithms for chorioamnionitis. Even at this current stage there are reasons to believe that proteomic biomarkers will improve the understanding of how chorioamnionitis programs or affects the fetus in utero, thus defining its exposome (sum of interactions between genetic make-up of the fetus and the intrauterine environment) of pregnancies affected by infection and/or inflammation. This review summarizes the results of proteomics studies that have aimed or reached these goals.

Figure 1

Flow chart representation of the phases and patient groups that led to the discovery and validation of the amniotic fluid proteomics mass-restricted (MR) score. PTL, preterm labor; PTB, preterm birth; PPROM, preterm premature rupture of membranes; AF, amniotic fluid; WBC, white blood cell; EONS, early onset neonatal sepsis. Flow chart sums subjects from references and .

Figure 2

(A) Representative surface-enhanced-laser desorption/ionization–time of flight mass spectrometry profiles of the amniotic fluid based on the increasing severity of inflammation. P1–P4 represent the biomarker peaks of the mass-restricted (MR) score; (B) Kaplan–Meier analysis illustrating the duration from amniocentesis to delivery in women with MR scores of 0 (zero, no inflammation), MR scores of 1–2 (mild inflammation), and MR scores of 3–4 (severe inflammation). IAI, intra-amniotic infection/inflammation. Published in PLoS Med 2007;4:e18. Republication by authors permitted under Creative Commons Attribution License. [Ref. 29]

Figure 3

Three-dimensional representation of the relationship between the amniotic fluid mass-restricted (MR) score and histological inflammatory findings in the fetal membranes. (A) Stages of chorioamnionitis; (B) grades of chorio-deciduitis; and (C) grades of amnionitis. With permission from: Obstet Gynecol 2008;111(2 Pt 1):403–12. KWH permission to republish 2766550305749. [Ref. 33]

Figure 4

Morphological (Masson trichrome, A and C) and immunohistochemical (B and D) staining of umbilical cord affected by severe histological funisitis. (A and B) Neutrophils infiltrating the wall of an umbilical artery (UA) and perivascular Wharton gelatin stain positive for calgranulin C, one of the biomarkers of the MR score. (C and D) Neutrophils adhering to the endothelium of the umbilical vein (UV) intensely positive for calgranulin C. With permission from: Am J Obstet Gynecol 2007;196:181.e1–13. Elsevier permission to republish 2765421478702. [Ref. 48]

Figure 5

Flow chart representation of the phases and patient groups used for discovery and validation of the cord blood biomarkers of antenatal exposure to intra-amniotic infection/inflammation. PTL, preterm labor; PTB, preterm birth; PPROM, preterm premature rupture of membranes; NBSCU, NewBorn Special Care Unit; CB, cord blood; EONS, early onset neonatal sepsis; HCA, histological chorioamnionitis; IVH, intraventricular hemorrhage; ROP, retinopathy of prematurity; NEC, necrotizing enterocolitis, BPD, bronchopulmonary dysplasia; 2D-DIGE, two-dimensional differential gel electrophoresis. Published in PLoS One 2011;6:e18. Republication by authors permitted under Creative Commons Attribution License. [Ref. 29]

Figure 6

Merged two-dimensional differential gel electrophoresis image of one of the three gels used during the proteomics discovery phase and representative spots identified by mass spectrometry. Spots upregulated at least 1.5-fold are outlined in blue and spots downregulated at least 1.5-fold are outlined with red. The squared regions are shown with matched unambiguous protein identities and direction of change represented by blue arrows. Apo, apolipoprotein; Hp, haptoglobin; HpRP, haptoglobin-related protein. Published in PLoS One 2011;6:e18. Republication by authors permitted under Creative Commons Attribution License. [Ref. 29]

Figure 7

Cord blood haptoglobin (Hp) and haptoglobin-related protein (HpRP) immunoreactivity revealed by western blot and enzyme-linked immunosorbent assay (ELISA). (A) Western blot of umbilical vein (UV) serum from four preterm newborns of similar gestational age at birth. Newborns in lanes 1–3 were diagnosed with early onset neonatal sepsis (EONS) based on clinical manifestations and hematological indices (presumed EONS) and all received intravenous antibiotics. The newborn in lane 4 had a negative sepsis work-up. Blood cultures remained negative for all four newborns. The presence of a conspicuous immunoreactive band corresponding to the â-chain (~42 kDa) in lanes 1–3 is consistent with our defined switched-on Hp pattern. The absence of this band indicates for a switched-off pattern in lane 4. The band ~9 kDa (lanes 1 and 2) corresponds to the á1-chain whereas the band at ~20 kDa (lanes 2–3) corresponds to the á2-chain. (B) Impact of Hp phenotypes on Hp&HpRP immunoreactivity as measured by ELISA. The red line indicates the group’s median. Groups assigned different letters are statistically different at P < 0.05 (Kruskal–Wallis analysis of variance). (C) Western blot of three representative maternal (Mat) and UV sera retrieved from women with normal term deliveries. Note the switched-off pattern of the cord blood in contrast to the switched-on pattern of the adult blood. Published in PLoS One 2011;6:e18. Republication by authors permitted under Creative Commons Attribution License. [Ref. 29]

Figure 8

Clustering algorithm based on probability of ‘antenatal exposure to intra-amniotic infection/inflammation (IAI)’. Posterior probabilities of antenatal exposure were calculated for all eight possible combinations of significant indicators [cord blood haptoglobin (Hp) switch pattern, cord blood interleukin (IL)-6, presumed early onset neonatal sepsis (EONS)] and their modal characteristics. The number of newborns presenting each combination is included in parentheses. Published in PLoS One 2011;6:e18. Republication by authors permitted under Creative Commons Attribution License. [Ref. 29]