Abstract
Objectives:
1) To characterize the cellular composition of the amniotic fluid of patients diagnosed with clinical chorioamnionitis at term, as a function of the presence or absence of microorganisms determined by cultivation techniques, and 2) to characterize the cytokine production by white blood cells present in the amniotic fluid using flow cytometry-based techniques.
Materials and methods:
Amniotic fluid samples from 20 women who had the diagnosis of clinical chorioamnionitis at term were analyzed using cultivation techniques (for aerobic and anaerobic bacteria as well as genital Mycoplasmas). Amniotic fluid IL-6 concentrations were determined by an enzyme-linked immunosorbent assay. Amniotic fluid leukocytes were visualized by using hematoxylin and eosin staining and immunofluorescence. Immunophenotyping of surface markers and cytokines was performed in amniotic fluid leukocytes using flow cytometry.
Results:
1) Neutrophils (CD45+CD15+ cells) were the most common leukocyte subset found in the amniotic fluid, followed by monocytes (CD45+CD14+ cells); other white blood cells (such as lymphocytes and natural killer cells) were scarce in the amniotic fluid; 2) the absolute counts of neutrophils and monocytes were significantly higher in patients with microorganisms found in the amniotic fluid than in those without detectable microorganisms, using cultivation techniques; 3) there was a significant correlation between the absolute counts of neutrophils and monocytes determined by flow cytometry (Spearman’s correlation=0.97; P<0.001); 4) there was a significant correlation between the absolute white blood cell count determined with a hemocytometer chamber and by flow cytometric analysis (Spearman’s correlation=0.88; P<0.001); and 5) the profile of cytokine expression differed between monocytes and neutrophils; while neutrophils predominantly produced TNF-α and MIP-1β, monocytes expressed higher levels of IL-1β and IL-1α.
Conclusion:
Flow cytometry analysis of the amniotic fluid of patients with intra-amniotic infection and clinical chorioamnionitis at term demonstrated that neutrophils and monocytes are the most common cells participating in the inflammatory process. We have characterized, for the first time, the differential cytokine expression by these cells in this important complication of pregnancy.
Introduction
Clinical chorioamnionitis at term is the most common diagnosis related to infection made in labor and delivery units worldwide [1], [2], [3], [4], [5], [6], [7], [8]. This term refers to a clinical syndrome traditionally attributed to microbial invasion of the amniotic cavity frequently caused by genital mycoplasmas, Gardnerella vaginalis, and other commensal organisms observed in the lower genital tract [9], [10]. Microorganisms and their products can elicit a local inflammatory response in the amniotic cavity [11], [12], [13], [14], [15], [16], [17], termed intra-amniotic inflammation [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], which may extend to two hosts – the fetus [28], [29], [30], [31] and the mother [32]. Clinical chorioamnionitis represents evidence of a systemic maternal inflammatory response [32] but not necessarily a fetal inflammatory response [31].
The intra-amniotic inflammatory response is characterized by an increased amniotic fluid white blood cell count [33], [34], [35], [36], [37] and increased concentrations of inflammatory mediators such as cytokines [27], [34], [35], [36], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50], [51], [52], [53], [54], [55], [56], [57], [58], [59], [60], [61], [62], [63], [64], [65], [66], [67], [68], [69], [70], [71], [72], [73], [74], [75] and prostaglandins [76], [77], [78], [79], [80], [81], [82], [83], [84], [85]. However, there is a paucity of information about the cellular composition of the intra-amniotic inflammatory response in clinical chorioamnionitis, and most studies have reported changes in the total white blood cell count of the amniotic fluid while using a hemocytometer chamber (e.g. a Neubauer chamber).
Flow cytometry has emerged as a state-of-the-art procedure to assess the phenotypic characteristics of cells participating in inflammatory responses [86], [87], [88], [89]. This technique characterizes their functional state (i.e. assesses reactive oxygen species and cytokine production) [90], [91], [92], [93], [94], determines viability [95], [96], [97], and evaluates other important biological characteristics [98], [99], [100], [101], [102], [103].
The purpose of this study was to characterize by flow cytometry the cellular composition of the amniotic fluid of patients diagnosed with clinical chorioamnionitis at term, as a function of the presence or absence of microorganisms determined by conventional cultivation techniques. Moreover, this study was designed to characterize cytokine production by white blood cells present in the amniotic fluid by using flow cytometry-based techniques.
Materials and methods
Study population
This was a cross-sectional study of patients diagnosed with clinical chorioamnionitis at term who underwent transabdominal amniocentesis to identify microorganisms in the amniotic cavity. Patients were enrolled at Hutzel Women’s Hospital of the Detroit Medical Center (November 2013–July 2015). Inclusion criteria were: 1) singleton gestation; 2) gestational age ≥37 weeks; and 3) sufficient amniotic fluid obtained by transabdominal amniocentesis for immunophenotyping using flow cytometry. In all cases, amniocenteses were performed for clinical indications.
Maternal and neonatal data were obtained from retrospective clinical chart reviews; the information included: 1) the use of epidural analgesics and intrapartum antibiotics, 2) the membrane status (intact or ruptured) at the time of amniocentesis, and 3) the mode of delivery. Patients diagnosed with clinical chorioamnionitis (see diagnostic criteria below) were counseled by their treating physicians about the potential value of knowing the precise microorganism(s) involved in the suspected infection. Further management of these patients was at the discretion of the attending physician.
All patients provided written informed consent to donate additional amniotic fluid for research purposes, according to protocols approved by the Institutional Review Boards of Wayne State University and NICHD.
Clinical definitions
Gestational age was determined by the date of the last menstrual period and confirmed by ultrasound examination. The gestational age derived from sonographic fetal biometry was used if the estimation was inconsistent with menstrual dating. Clinical chorioamnionitis was diagnosed by the presence of maternal fever (temperature >37.8°C) accompanied by two or more of the following criteria: 1) uterine tenderness; 2) malodorous vaginal discharge; 3) fetal tachycardia (heart rate >160 beats/min); 4) maternal tachycardia (heart rate >100 beats/min); and 5) maternal leukocytosis (leukocyte count >15,000 cells/mm3) [2], [3], [4], [6]. Spontaneous term labor was defined as the presence of regular uterine contractions with a frequency of at least 1 every 10 min and cervical change after 37 weeks of gestation.
Microbial invasion of the amniotic cavity was defined according to the results of the amniotic fluid cultures [104], [105], [106], [107], [108]. Intra-amniotic inflammation was diagnosed when the amniotic fluid interleukin-6 (IL-6) concentration was ≥2.6 ng/mL [51], [109]. Based on the results of the amniotic fluid cultures, patients were classified as having either a negative or a positive culture. Acute histologic chorioamnionitis was diagnosed based on the presence of inflammatory cells in the chorionic plate and/or chorioamniotic membranes [110], [111], [112], [113], while acute funisitis was diagnosed by the presence of neutrophils in the wall of the umbilical vessels and/or Wharton’s jelly, using criteria previously described [110], [113], [114], [115], [116], [117].
Sample collection
Amniotic fluid was retrieved by transabdominal amniocentesis, which was performed under antiseptic conditions, using a 22-gauge needle monitored by ultrasound. Each amniotic fluid sample was transported to the clinical laboratory in a capped sterile syringe and cultured for aerobic and anaerobic bacteria as well as for genital mycoplasmas [10], [33], [34], [35], [36], [37], [48], [118], [119], [120]. Shortly after collection, a white blood cell count was performed for each amniotic fluid sample using a hemocytometer chamber, according to methods previously described [33]. Glucose concentrations [121] were determined and Gram stains [122], [123] were performed in the amniotic fluid samples.
Determination of IL-6 in amniotic fluid
Concentrations of IL-6 in the amniotic fluid were determined by using a sensitive and specific enzyme immunoassay obtained from R&D Systems (Minneapolis, MN, USA). The IL-6 concentrations were determined by interpolation from the standard curves. The inter- and intra-assay coefficients of variation for IL-6 were 8.7% and 4.6%, respectively. The detection limit of the IL-6 assay was 0.09 pg/mL. The IL-6 concentrations in the amniotic fluid were determined for clinical purposes. It has been previously reported that IL-6 concentrations are useful for the detection of intra-amniotic inflammation [51], [109].
Hematoxylin and eosin staining
Amniotic fluid cells were counted using an automatic cell counter (Cellometer Auto 2000; Nexcelom, Lawrence, MA, USA). Amniotic fluid cells (5×104) were cytospun onto a Fisherbrand Superfrost Plus microscope slide (Thermo Fisher Scientific, Rochester, NY, USA) using a Shandon Cytospin 3 cytocentrifuge (Thermo Fisher Scientific) at 800 rpm for 5 min. The slide was then stained with hematoxylin (catalog number 88018, Thermo Scientific) for 10 s, washed with distilled water, immersed in 80% ethanol, and stained with eosin (catalog number 71211, Thermo Scientific) for 10 s. Next, each slide was dehydrated by subsequent washes with 95% ethanol, 100% ethanol, and xylene, and mounted with xylene. Images were obtained using a Pannoramic MIDI slide scanner (PerkinElmer, Waltham, MA, USA).
Immunofluorescence
Amniotic fluid cells (5×104) were cytospun onto a Fisherbrand Superfrost Plus microscope slide (Thermo Fisher Scientific) using a Shandon Cytospin 3 cytocentrifuge (Thermo Fisher Scientific) at 800 rpm for 5 min. The cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA), and washed with 1× phosphate-buffered saline (1× PBS; Life Technologies, Grand Island, NY, USA). Non-specific antibody interaction was blocked using Dako Protein Block Serum-Free Solution (catalog number X0909; DakoCytomation, Carpinteria, CA, USA) for 30 min at room temperature. Next, cells were incubated with mouse anti-human CD14 (clone MϕP9, catalog number 347490, BD Biosciences, San Jose, CA, USA) for 1 h at room temperature. Cells were then washed with 1× PBS containing 0.1% Tween 20 (PBST) (Sigma-Aldrich, St. Louis, MO, USA). After blocking for 10 min with 10% goat serum (KPL, Gaithersburg, MD, USA), a secondary goat anti-mouse IgG-Alexa Fluor 594 (catalog number A11032; Life Technologies) was added and cells were incubated for 1 h at room temperature in the dark. Following incubation, cells were washed with PBST and were incubated with FITC-conjugated mouse anti-human CD15 (clone W6D3, catalog number 5623790, BD Biosciences) for 1 h at room temperature in the dark. Finally, cells were washed with PBST and then mounted using ProLong Diamond Antifade Mountant with DAPI (Life Technologies). Immunofluorescence was visualized using an Olympus BX60 fluorescence microscope (Olympus, Tokyo, Japan). Images were obtained using an Olympus DP71 camera and DP Controller Software (Olympus).
Immunophenotyping of amniotic fluid leukocytes by flow cytometry
Amniotic fluid samples (1.5 mL) were washed with 1 mL of 1× PBS and the resulting cell pellet was re-suspended in 1 mL of 1× PBS. One microliter of the Fixable Viability Stain 510 reagent (catalog number 564406; BD Biosciences) was added to the cell suspension, which was incubated for 30 min at 4°C in the dark. Following incubation, the cell suspension was centrifuged at 285×g and the cell pellet was washed with 1 mL of FACS buffer [BSA 0.1% (Sigma-Aldrich)], sodium azide 0.05% (Fisher Scientific Bioreagents, Fair Lawn, NJ, USA), and 1× PBS)]. The cell pellet was then incubated with 10 μL of human FcR blocking reagent (Miltenyi Biotec, San Diego, CA, USA) in 40 μL of stain buffer (catalog number 554656; BD Biosciences) for 10 min at 4°C. Next, the cells were incubated with the following extracellular fluorochrome-conjugated anti-human antibodies (BD Biosciences) for 30 min at 4°C in the dark: CD45-Alexa Fluor 700 (clone HI30), CD15-Brilliant Violet 650 (clone HI98), CD14-Brilliant Ultraviolet 395 (clone MϕP9), CD19-PE-Cy5 (clone HIB19), CD3-APC-Cy7 (clone SK7), and CD56-Brilliant Violet 711 (clone NCAM18.2). After extracellular staining, the cells were washed with 1× PBS to remove excess antibody, re-suspended in 0.5 mL of stain buffer, and acquired using the BD LSRFortessa flow cytometer (BD Biosciences) and BD FACSDiva 6.0 software (BD Biosciences). The analysis and figures were performed using the FlowJo software version 10 (FlowJo, Ashland, OR, USA). Neutrophils and monocytes were defined as CD45+CD15+ cells and CD45+CD14+ cells, respectively. The total number of neutrophils and monocytes were determined using CountBright absolute counting beads (Molecular Probes, Eugene, OR, USA).
Cytokine expression assessed by flow cytometry
Cytokine expression was assessed by intracellular staining. Following extracellular staining, the cells were fixed and permeabilized using BD Cytofix/Cytoperm Fixation and Permeabilization Solution (BD Biosciences). Next, the cells were washed with 1× BD Perm/Wash Buffer (BD Biosciences), re-suspended in 50 μL of the same buffer, and stained with the following intracellular antibodies (BD Biosciences and Biolegend, San Diego, CA, USA) for 30 min at 4°C in the dark: macrophage inflammatory protein (MIP)-1α-PE (clone 11A3), MIP-1β-PerCP-Cy5.5 (clone D21-1351), IL-1α-FITC (clone A55), IL-1β-Alexa Fluor 647 (clone JK1B-1), IL-8-Brilliant Violet 421 (clone G265-8), and TNF-α-Brilliant Violet 605 (clone Mab11). Isotype controls were also prepared. Finally, the stained cells were washed with 1× BD Perm/Wash Buffer, re-suspended in 0.5 mL stain buffer, and acquired using the BD LSR II and LSRFortessa flow cytometers and BD FACSDiva 6.0 software. The analysis was performed using the FlowJo software version 10. The mean fluorescence intensity (MFI) of every cytokine was calculated as [MFI (anti-cytokine mAb) – MFI (isotype control mAb)].
Statistical analysis
Statistical analysis was performed using the R package [124]. Normality of the data was tested using the Wilk-Shapiro test. Mann-Whitney U-tests were used to compare the medians among the groups. Comparisons of the proportions were made using Fisher’s exact tests. Spearman’s correlation was used to examine the relationship between continuous variables. A P-value of <0.05 was used to determine statistical significance.
Results
Characteristics of the study population
A total of 20 patients diagnosed with clinical chorioamnionitis between 37 and 42 weeks of gestation were included in this study. Demographic and clinical characteristics of the study population are displayed in Table 1. Seven patients had amniotic fluid cultures negative for bacteria, and 13 patients had positive cultures. There were no significant differences in the median maternal age, frequency of nulliparity, pre-pregnancy body mass index, gestational age at amniocentesis and at delivery, frequency of vaginal delivery, and birthweight among the groups.
Clinical characteristics of the study population.
| Negative amniotic fluid culture (n = 7) | Positive amniotic fluid culture (n =13) | P-value | |
|---|---|---|---|
| Maternal age (years) | 25 (23–29) | 21 (20–24) | 0.08 |
| Nulliparity | 57.1% (4/7) | 76.9% (10/13) | 0.62 |
| Body mass index (kg/m2) | 35.7 (32.1–36.5) | 29.7 (25.225–30.7) | 0.06 |
| Gestational age at amniocentesis (weeks) | 39.3 (38.9–39.8) | 40.3 (38.1–40.9) | 0.15 |
| Rupture of membranes prior to amniocentesis | 28.6% (2/7) | 92.3% (12/13) | 0.008 |
| Epidural before amniocentesis | 28.6% (2/7) | 92.3% (12/13) | 0.008 |
| Conventional amniotic fluid analysis | |||
| – Red blood cell count (cells/mm3) | 4 (4–131.5) | 328 (128–2599) | 0.02 |
| – White blood cell count (cells/mm3) | 1 (0–8.5) | 655 (19–1445) | 0.01 |
| – Glucose (mg/dL) | 11 (4.5–17) | 1 (1–1) | 0.002 |
| – Interleukin-6 (ng/mL) | 1.1 (0.38–3.48) | 13.2 (9–50.3) | 0.02 |
| Gestational age at delivery (weeks) | 39.7 (39.3–40.1) | 40.4 (38.1–40.9) | 0.38 |
| Vaginal delivery | 71.4% (5/7) | 61.5% (8/13) | 1 |
| Birthweight (g) | 3455 (3350–3687.5) | 3445 (3000–3745) | 0.70 |
| Acute histologic chorioamnionitis | 71.4% (5/7) | 92.3% (12/13) | 0.49 |
| Acute funisitis | 28.6% (2/7) | 92.3% (12/13) | 0.008 |
Data presented as median (interquartile range) or % (n).
Patients with amniotic fluid cultures positive for microorganisms had a higher frequency of ruptured membranes than those with negative amniotic fluid cultures [92.3% (12/13) vs. 28.6% (2/7); P=0.008]. Patients with positive amniotic fluid cultures had significantly higher median amniotic fluid total white blood cell counts and IL-6 concentrations, as well as lower amniotic fluid glucose concentrations, than women with negative amniotic fluid cultures.
Funisitis was more common in women with positive amniotic fluid cultures than in those with negative amniotic fluid cultures [92.3% (12/13) vs. 28.6% (2/7), P=0.008].
Table 2 describes microorganisms in the amniotic fluid inflammatory response as well as white blood cell counts determined by flow cytometry in patients with positive amniotic fluid cultures.
Amniotic fluid and placental characteristics of patients with clinical chorioamnionitis at term who had positive amniotic fluid cultures.
| Case number | Gestational age at amniocentesis (weeks) | Clinical laboratory | Flow cytometry | Microorganisms in amniotic fluid | Acute histologic chorioamnionitisa | Acute funisitisb | ||
|---|---|---|---|---|---|---|---|---|
| Total amniotic fluid white blood cell count (cells/mm3) | Amniotic fluid IL-6 (ng/mL) | Neutrophils (CD15 cells/mm3) | Monocytes (CD14 cells/mm3) | |||||
| 1 | 37.9 | 864 | 1.91 | 1427.2 | 48.1 | Gardnerella vaginalis, Mycoplasma hominis, Peptostreptococcus, Streptococcus viridans, Ureaplasma urealyticum, Porphyromonas | Stage 2 | Stage 1 |
| 2 | 41.3 | 19 | 41.6 | 18.4 | 0.3 | Ureaplasma urealyticum, Streptococcus anginosus,Beta hemolytic Streptococcus | Stage 2 | Stage 1 |
| 3 | 37.1 | 1445 | 131.8 | 647.2 | 10.9 | Mycoplasma hominis, Streptococcus group B, Ureaplasma urealyticum | Stage 2 | Stage 1 |
| 4 | 41.6 | 655 | 50.3 | 193.1 | 6.5 | Mycoplasma hominis, Ureaplasma urealyticum | Stage 2 | Stage 1 |
| 5 | 40.9 | 2053 | 13.2 | 547.6 | 20.4 | Lactobacillus Jensenii | Stage 2 | Stage 1 |
| 6 | 40.6 | 0 | 1.39 | 0.4 | 0.0 | Mycoplasma hominis, Ureaplasma urealyticum | Stage 1 | Stage 1 |
| 7 | 40.3 | 790 | 18.4 | 513.3 | 10.9 | Gardnerella vaginalis, Actinomyces israelii, Enterococcus faecalis | Stage 2 | Stage 2 |
| 8 | 39.9 | 0 | 1.49 | 0.0 | 0.0 | Streptococcus group B | Stage 2 | Stage 2 |
| 9 | 38.1 | 70 | 11.48 | 28.1 | 2.4 | Ureaplasma urealyticum | None | None |
| 10 | 38 | 2090 | 9 | 210.7 | 8.4 | Ureaplasma urealyticum | Stage 2 | Stage 1 |
| 11 | 40.9 | 2600 | 51.1 | 543.8 | 9.3 | Ureaplasma urealyticum, Candida albicans, Candida Glabrata, Candida tropicalis, Klebsiella | Stage 1 | Stage 1 |
| 12 | 40.1 | 9 | 10.5 | 4.5 | 0.1 | Streptococcus group B | Stage 2 | Stage 1 |
| 13 | 41.1 | 340 | 51.3 | 141.4 | 2.2 | Ureaplasma urealyticum | Stage 2 | Stage 1 |
aAcute histologic chorioamnionitis: Stage 1, early, acute subchorionitis/chorionitis; Stage 2, intermediate, acute chorioamnionitis.
bFunisitis: Stage 1, early, umbilical phlebitis/chorionic vasculitis; Stage 2, intermediate, umbilical arteritis.
Neutrophil and monocyte counts are higher in patients with positive amniotic fluid cultures than in those with negative amniotic fluid cultures
Figure 1 represents the gating strategy used to identify leukocytes in the amniotic fluid samples. Briefly, total leukocytes were identified using CD45 within the viability gate. Leukocytes expressing CD15 were considered to be neutrophils and those expressing CD14 were considered to be monocytes. Flow cytometry demonstrated that neutrophils and monocytes were the most abundant leukocyte subsets observed in the amniotic fluid of patients with clinical chorioamnionitis at term and positive amniotic fluid cultures (Figure 1). Lymphocytes (CD45+CD3+ and CD45+CD19+ cells) and natural killer cells (CD45+CD56+ cells) were scarce in the amniotic fluid samples. Hematoxylin and eosin staining of the amniotic fluid exudate showed the typical morphology of neutrophils (N; green arrow) and monocytes (M; red arrow) (Figure 2A). Immunofluorescence of these amniotic fluid cells also revealed that neutrophils (CD15+ cells are green) and monocytes (CD14+ cells are red) are abundant in the amniotic fluid of patients with clinical chorioamnionitis at term (Figure 2B).
Gating strategy used to identify amniotic fluid leukocytes in women with clinical chorioamnionitis at term. Total leukocytes (CD45+ cells) were gated using singlets, FSC vs. SSC and within the viability gate. Neutrophils (CD15+ cells) and monocytes (CD14+ cells) were gated within the CD45+ gate.
Histological examination of amniotic fluid neutrophils and monocytes in women with clinical chorioamnionitis at term. (A) Hematoxylin and eosin staining shows the typical morphology of neutrophils (green arrow) and monocytes (red arrow) in the amniotic fluid of women with clinical chorioamnionitis at term. Magnification 400×. Scale bars: 50 μm. (B) Immunofluorescence of neutrophils and monocytes in the amniotic fluid of women with clinical chorioamnionitis at term. Neutrophils (CD15+ cells) are green, monocytes (CD14+ cells) are red, and nuclei are blue (DAPI). Magnification 400×.
The median neutrophil count was higher in patients with amniotic fluid cultures positive for microorganisms than in those with negative amniotic fluid cultures [193.1 cells/mm3 (18.4–543.8) vs. 0.4 cells/mm3 (0.1–8.2); P=0.01] (Figure 3). Similarly, the median monocyte count was significantly higher in those with amniotic fluid cultures positive (vs. negative) for microorganisms [6.5 cells/mm3 (0.3–10.9) vs. 0 cells/mm3 (0.0–0.4); P=0.01] (Figure 3). There was a significant positive correlation between the amniotic fluid total white blood cell counts determined by flow cytometry and a hemocytometer chamber (Spearman’s correlation=0.88; P<0.001; Table 3, Figure 4). There was a significant positive correlation between the amniotic fluid neutrophil and monocyte counts determined by flow cytometry (Spearman’s correlation=0.97; P<0.001; Table 3). There were significant positive correlations between IL-6 concentrations and the amniotic fluid neutrophil (Spearman’s correlation=0.59; P=0.007) and monocyte (Spearman’s correlation=0.61; P=0.004) counts determined by flow cytometry (Table 3).
Total number of amniotic fluid neutrophils and monocytes in women with clinical chorioamnionitis at term. Total number of neutrophils and monocytes in women with clinical chorioamnionitis at term with negative and positive cultures. Data are shown as scatter plots (median). Mann-Whitney U-tests.
The relationship of the white blood cell counts between flow cytometry and the standard techniques, as well as the amniotic fluid IL-6 concentrations, in patients with clinical chorioamnionitis at term.
| Correlation between | Correlation coefficient (Spearman correlation) | P-value |
|---|---|---|
| Total white blood cell count (cells/mm3) by hemocytometer and total white blood cell count by flow cytometry | 0.88 | <0.001 |
| Neutrophils (CD15 cells/mm3) and monocytes (CD14 cells/mm3) by flow cytometry | 0.97 | <0.001 |
| Amniotic fluid Interleukin-6 concentration (ng/mL) and | ||
| – Neutrophils (CD15 cells/mm3) by flow cytometry | 0.59 | 0.007 |
| – Monocytes (CD14 cells/mm3) by flow cytometry | 0.61 | 0.004 |
| – Total white blood cell count (cells/mm3) by flow cytometry | 0.59 | 0.007 |
Scatter plot of Spearman’s correlation between the amniotic fluid white blood cell counts determined by a hemocytometer and those determined by flow cytometry in patients with clinical chorioamnionitis at term. Black line indicates linear regression line.
The organisms involved, as well as the number of neutrophils and monocytes, are described in Table 2. The most common microorganism was the Ureaplasma species, followed by Streptococcus agalactiae and Gardnerella vaginalis.
Cytokine expression by neutrophils and monocytes
Cytokine expression by the amniotic fluid white blood cells was studied in patients with positive amniotic fluid cultures. Patients with clinical chorioamnionitis but negative amniotic fluid cultures did not have enough cells in 1.5 mL to study cytokine expression. Neutrophils had a higher median mean fluorescence intensity (MFI) for TNF-α and MIP-1β than monocytes (Figure 5A and B). In contrast, monocytes had a higher median MFI for IL-1α and IL-1β than neutrophils (Figure 5C and D). The median MFI for IL-8 and MIP-1α was not significantly different between neutrophils and monocytes (Figure 5E and F).
![Figure 5: Cytokine expression by amniotic fluid neutrophils and monocytes in women with clinical chorioamnionitis at term and positive cultures. The mean fluorescence intensity (MFI) of TNF-α (A), MIP-1β (B), IL-1α (C), IL-1β (D), IL-8 (E) and MIP-1α (F) was calculated as [MFI (anti-cytokine mAb) – MFI (isotype control mAb)]. Data are shown as scatter plots (median). Mann-Whitney U-tests.](/document/doi/10.1515/jpm-2016-0225/asset/graphic/j_jpm-2016-0225_fig_005.jpg)
Cytokine expression by amniotic fluid neutrophils and monocytes in women with clinical chorioamnionitis at term and positive cultures. The mean fluorescence intensity (MFI) of TNF-α (A), MIP-1β (B), IL-1α (C), IL-1β (D), IL-8 (E) and MIP-1α (F) was calculated as [MFI (anti-cytokine mAb) – MFI (isotype control mAb)]. Data are shown as scatter plots (median). Mann-Whitney U-tests.
Discussion
Principal findings of the study
The study herein is a novel report about the cytometric characteristics of the amniotic fluid of patients diagnosed with clinical chorioamnionitis at term. The principal findings of the study are: 1) neutrophils (CD45+CD15+ cells) were the most common leukocyte subset found in the amniotic fluid, followed by monocytes (CD45+CD14+ cells); other white blood cells (such as lymphocytes and natural killer cells) were scarce in the amniotic fluid; 2) the absolute counts of neutrophils and monocytes were significantly higher in patients with detectable microorganisms in the amniotic fluid than in those without detectable microorganisms, using cultivation techniques; 3) there was a significant correlation between the absolute counts of neutrophils and monocytes, determined by flow cytometry (r=0.97; P<0.001), and also between the absolute white blood cell count determined with a hemocytometer chamber and the counts derived from flow cytometric analysis (r=0.88; P<0.001); and 4) the profile of cytokine expression was different for monocytes and neutrophils; while neutrophils predominantly expressed TNF-α and MIP-1β, monocytes expressed higher levels of IL-1α and IL-1β. Collectively, these findings suggest that the inflammatory exudate in intra-amniotic inflammation is complex and that neutrophils and monocytes play different roles in clinical chorioamnionitis at term.
Flow cytometric analysis of amniotic fluid
The standard method to enumerate white blood cells in the amniotic fluid utilizes a hemocytometer chamber (e.g. a Neubauer chamber) [33], [34], [35], [36], [37], [125] and a cytospin to perform a differential count [33]. Flow cytometry allows the identification of cell populations using specific surface markers and also the examination of functional properties such as cytokine production [86], [87], [88], [89], [90], [91], [92], [93], [126]. The present study was undertaken to gain insights into the nature of the inflammatory response in clinical chorioamnionitis at term and also to study, for the first time, the cytokine profile of leukocytes involved in intra-amniotic infection. The observation that neutrophils are the cells most commonly found in intra-amniotic infection is consistent with previous studies [33]. The fact that monocytes are the second most common cell is also consistent with prior observations [33].
Amniotic fluid neutrophils
Neutrophils are the most abundant leukocytes in the circulatory system and form the first line of defense against invading pathogens; they possess an arsenal of weapons suited for elimination of microbes [127]. These innate immune cells are first and foremost phagocytes, capable of enveloping and digesting microbes through the generation of reactive oxygen species (ROS) and NADPH-derived oxidants [128]. Neutrophils are granulocytic, carrying granules filled with a variety of enzymes that can be injected into the phagosome or released externally [127], [128]. Along with degranulation, neutrophils release a variety of antimicrobial proteins (such as defensins) and reactive nitrogen intermediates [128]. Recently, a new mechanism was described whereby neutrophils undergo a specialized cell death, releasing web-like structures composed of DNA, histones, and antimicrobial products such as neutrophil elastase [129]. Termed “neutrophil extracellular traps,” or NETs, this phenomenon represents the final containment effort of a neutrophil to combat microbial invasion [129]. Detection of invading microbes is largely dependent on the expression of specialized surface receptors and ligands. The carbohydrate epitope 3-fucosyl-N-acetyl-lactosamine (CD15), a classical marker for neutrophil identification, is highly expressed on activated neutrophils [130]. Neutrophils are constantly circulating and require a finely tuned homing and adhesion system in order to reach sites of infection. Expression of CXCR-1 and CXCR-2 allows neutrophils to detect the potent neutrophil-specific chemokine, IL-8 [131], while L-selectin [132], [133] and platelet endothelial cell adhesion molecule-1 [134] enable neutrophil adhesion and transmigration [128]. Along with antimicrobial mechanisms, neutrophils release cytokines in order to signal local and immune cells. Neutrophils primarily secrete pro-inflammatory cytokines such as TNF-α, IL-12, IL-1α, and IL-1β [135]. As first responders, neutrophils release multiple chemokines (IL-8, MIP-1α, and MIP-1β) to attract other immune cells as well as growth factors such as granulocyte-colony stimulating factor, vascular endothelial growth factor, and hepatocyte growth factor [135].
Neutrophils are the most abundant leukocytes found in the amniotic fluid in cases with intra-amniotic infection/inflammation [33]. Their fetal origin was observed when X and Y chromosomes were detected by fluorescence in situ hybridization in the amniotic fluid leukocytes of four women with intra-amniotic infection who delivered premature male infants [136]. Since the neutrophils seen in the chorioamniotic membranes are of maternal origin and the chorionic plate contains a mix of maternal and fetal neutrophils, it has been suggested that the fetal vasculature of the chorionic plate is the main source of the amniotic fluid neutrophils [137]. Yet, a more comprehensive study that includes a large sample size of cases presenting with intra-amniotic infection will be necessary in order to be certain of the fetal origin of the amniotic fluid neutrophils. We do not exclude the possibility, in some cases, that maternal leukocytes invade the amniotic cavity, given observations we have made, in which there is a severe maternal inflammatory response: a high amniotic fluid white blood cell count without evidence of a fetal inflammatory response (i.e. funisitis or chorionic vasculitis).
The mechanisms responsible for neutrophil chemotaxis into the amniotic fluid probably rely on the production of neutrophil chemokines, such as IL-8 [59], [60], [61], [62], [138], GROα [66], [67], and CXCL6 (granulocyte chemotactic protein-2) [63]. The fact that amniotic fluid neutrophils overexpressed TNF-α and MIP-1β suggests that their role is different from that of other inflammatory cells. TNF-α has been implicated in the mechanisms of membrane rupture through the production of matrix metalloproteinases as well as in the induction of amniotic cell death [56], [57], [139], [140], [141], [142]. MIP-1β is a monocyte chemokine and may therefore play a role in attracting neutrophils and monocytes in cases of intra-amniotic infection associated with bacteria [27]. IL-1α and IL-1β are also produced by neutrophils and have been implicated in the mechanisms of membrane rupture and parturition in general [38], [39], [143], [144].
Amniotic fluid monocytes
Monocytes are derived from a common myeloid progenitor in the bone marrow and are then released into the peripheral blood, where they circulate for several days before infiltrating tissues and becoming tissue macrophages [145]. Monocytes have multiple functions due to their lack of defined specialization. Monocytes primarily serve to supplement tissue-resident macrophage populations, increasing response strength and sheer numbers during infection or injury [146]. This function is enhanced by the splenic monocyte reservoirs that release stored monocytes in an angiotensin-II dependent manner [147]. Circulating monocytes are chemoattracted to both damaged and undamaged tissues where they undergo one of several fates [148], [149]. First, monocytes can be activated to a pro-inflammatory state through signals such as lipopolysaccharide and IFN-γ, clearing pathogens and releasing factors similar to classical macrophages such as TNF-α, IL-1β, IL-12, ROS, and NO intermediates through inducible nitric oxide synthase (iNOS) [146]. Alternatively, at non-infectious sites of injury, monocytes can acquire a repair-oriented phenotype through IL-4, IL-13, or IL-10 stimulation, increasing the release of homeostatic factors such as TGF-β [146]. Second, monocytes can replace decreased tissue-resident macrophage populations following injury-induced reduction, especially during infection [146], [148]. Third, in addition to macrophage population renewal and function, monocytes can also differentiate into dendritic cells that migrate to the lymph nodes and other tissues [150]. Monocytes display morphological heterogeneity, including size variability, granularity, and nuclear morphology [146]. These innate cells were initially identified by their expression of large amounts of CD14 [148], [151], a co-receptor for bacterial lipopolysaccharide [152]. Differential expression of CD14 and CD16 [153] allowed monocytes to be separated into two subsets: CD14highCD16− and CD14lowCD16+ cells [151]. CD14highCD16− cells are known as classic monocytes [151], while CD14lowCD16+ cells resemble tissue macrophages [154]. Therefore, the differential level expression of the CD14 molecule is a useful tool to distinguish monocytes from macrophages. Besides the differential expression of surface markers, monocytes are morphologically different from macrophages; therefore, histological examination is useful to confirm the state of differentiation of these innate immune cells [146], [155].
The study herein demonstrated that amniotic fluid monocytes are abundant in patients with clinical chorioamnionitis at term and bacteria in the amniotic fluid. Our findings are consistent with a previous report, which detected monocytes in the amniotic fluid by using Wright staining [33]. We found that monocytes produce IL-1α, IL-1β, IL-8, MIP-1α, and MIP-1β. However, the predominant cytokines produced by monocytes, when compared to neutrophils, were IL-1α and IL-1β. These cytokines participate in the process of parturition [38], [39], [143], [144] and in the host response to intra-amniotic infection [19], [38], [39], [40], [41], [42], [50], [156], [157].
Strengths and limitations
This is the first study to characterize the phenotypic properties of leukocytes in intra-amniotic inflammation for patients diagnosed with clinical chorioamnionitis at term. These findings are novel and provide insight into the biology of the inflammatory response in the context of intra-amniotic infection. Future studies could examine other properties of neutrophils, such as the formation of NETs and the production of reactive oxygen radicals. The current studies are limited to patients at term, and future studies of patients who will undergo preterm labor can provide insights into the biology of intra-amniotic inflammation in preterm parturition.
Conclusions
Flow cytometry analysis of the amniotic fluid from patients with intra-amniotic infection and clinical chorioamnionitis at term demonstrated that neutrophils and monocytes are the most common cells participating in the inflammatory process. We have characterized for the first time the differential cytokine expression by these cells in this important complication of pregnancy.
Acknowledgments
This research was supported, in part, by the Perinatology Research Branch, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, U.S. Department of Health and Human Services (NICHD/NIH/DHHS), and, in part, with federal funds from the NICHD/NIH/DHHS under Contract No. HHSN275201300006C. This research was also supported by the Wayne State University Perinatal Initiative in Maternal, Perinatal and Child Health. We thank the physicians and nurses from the Center for Advanced Obstetrical Care and Research, and the research assistants from the PRB Clinical Laboratory, for their help in collecting human samples. We also thank staff members of the PRB Histology and Pathology Units for their examination of the pathological sections.
References
[1] Gibbs RS. Diagnosis of intra-amniotic infection. Semin Perinatol. 1977;1:71–7.Search in Google Scholar
[2] Gibbs RS, Castillo MS, Rodgers PJ. Management of acute chorioamnionitis. Am J Obstet Gynecol. 1980;136:709–13.10.1016/0002-9378(80)90445-7Search in Google Scholar
[3] Gilstrap LC, 3rd, Cox SM. Acute chorioamnionitis. Obstet Gynecol Clin North Am. 1989;16:373–9.10.1016/S0889-8545(21)00165-0Search in Google Scholar
[4] Newton ER. Chorioamnionitis and intraamniotic infection. Clin Obstet Gynecol. 1993;36:795–808.10.1097/00003081-199312000-00004Search in Google Scholar
[5] Rouse DJ, Landon M, Leveno KJ, Leindecker S, Varner MW, Caritis SN, et al. The maternal-fetal medicine units cesarean registry: chorioamnionitis at term and its duration–relationship to outcomes. Am J Obstet Gynecol. 2004;191:211–6.10.1016/j.ajog.2004.03.003Search in Google Scholar
[6] Tita AT, Andrews WW. Diagnosis and management of clinical chorioamnionitis. Clin Perinatol. 2010;37:339–54.10.1016/j.clp.2010.02.003Search in Google Scholar
[7] Fishman SG, Gelber SE. Evidence for the clinical management of chorioamnionitis. Semin Fetal Neonatal Med. 2012;17:46–50.10.1016/j.siny.2011.09.002Search in Google Scholar
[8] Mazaki-Tovi S, Vaisbuch E. Clinical chorioamnionitis – an ongoing obstetrical conundrum. J Perinat Med. 2016;44:1–4.10.1515/jpm-2015-0366Search in Google Scholar
[9] Gibbs RS, Blanco JD, St Clair PJ, Castaneda YS. Quantitative bacteriology of amniotic fluid from women with clinical intraamniotic infection at term. J Infect Dis. 1982;145:1–8.10.1093/infdis/145.1.1Search in Google Scholar
[10] Romero R, Miranda J, Kusanovic JP, Chaiworapongsa T, Chaemsaithong P, Martinez A, et al. Clinical chorioamnionitis at term I: microbiology of the amniotic cavity using cultivation and molecular techniques. J Perinat Med. 2015;43:19–36.10.1515/jpm-2014-0249Search in Google Scholar
[11] Gravett MG, Witkin SS, Haluska GJ, Edwards JL, Cook MJ, Novy MJ. An experimental model for intraamniotic infection and preterm labor in rhesus monkeys. Am J Obstet Gynecol. 1994;171:1660–7.10.1016/0002-9378(94)90418-9Search in Google Scholar
[12] Bennett WA, Terrone DA, Rinehart BK, Kassab S, Martin JN, Jr., Granger JP. Intrauterine endotoxin infusion in rat pregnancy induces preterm delivery and increases placental prostaglandin F2alpha metabolite levels. Am J Obstet Gynecol. 2000;182:1496–501.10.1067/mob.2000.106848Search in Google Scholar
[13] Kallapur SG, Willet KE, Jobe AH, Ikegami M, Bachurski CJ. Intra-amniotic endotoxin: chorioamnionitis precedes lung maturation in preterm lambs. Am J Physiol Lung Cell Mol Physiol. 2001;280:L527–36.10.1152/ajplung.2001.280.3.L527Search in Google Scholar
[14] Grigsby PL, Hirst JJ, Scheerlinck JP, Phillips DJ, Jenkin G. Fetal responses to maternal and intra-amniotic lipopolysaccharide administration in sheep. Biol Reprod. 2003;68:1695–702.10.1095/biolreprod.102.009688Search in Google Scholar
[15] Newnham JP, Shub A, Jobe AH, Bird PS, Ikegami M, Nitsos I, et al. The effects of intra-amniotic injection of periodontopathic lipopolysaccharides in sheep. Am J Obstet Gynecol. 2005;193:313–21.10.1016/j.ajog.2005.03.065Search in Google Scholar
[16] Berry CA, Nitsos I, Hillman NH, Pillow JJ, Polglase GR, Kramer BW, et al. Interleukin-1 in lipopolysaccharide induced chorioamnionitis in the fetal sheep. Reprod Sci. 2011;18:1092–102.10.1177/1933719111404609Search in Google Scholar
[17] Grigsby PL, Novy MJ, Sadowsky DW, Morgan TK, Long M, Acosta E, et al. Maternal azithromycin therapy for Ureaplasma intraamniotic infection delays preterm delivery and reduces fetal lung injury in a primate model. Am J Obstet Gynecol. 2012;207:475 e1–4.10.1016/j.ajog.2012.10.871Search in Google Scholar
[18] Gibbs RS, Romero R, Hillier SL, Eschenbach DA, Sweet RL. A review of premature birth and subclinical infection. Am J Obstet Gynecol. 1992;166:1515–28.10.1016/0002-9378(92)91628-NSearch in Google Scholar
[19] Gomez R, Ghezzi F, Romero R, Munoz H, Tolosa JE, Rojas I. Premature labor and intra-amniotic infection. Clinical aspects and role of the cytokines in diagnosis and pathophysiology. Clin Perinatol. 1995;22:281–342.10.1016/S0095-5108(18)30286-0Search in Google Scholar
[20] Goncalves LF, Chaiworapongsa T, Romero R. Intrauterine infection and prematurity. Ment Retard Dev Disabil Res Rev. 2002;8:3–13.10.1002/mrdd.10008Search in Google Scholar PubMed
[21] Keelan JA, Blumenstein M, Helliwell RJ, Sato TA, Marvin KW, Mitchell MD. Cytokines, prostaglandins and parturition–a review. Placenta. 2003;(24 Suppl A):S33–46.10.1053/plac.2002.0948Search in Google Scholar PubMed
[22] Romero R, Espinoza J, Goncalves LF, Kusanovic JP, Friel L, Hassan S. The role of inflammation and infection in preterm birth. Semin Reprod Med. 2007;25:21–39.10.1055/s-2006-956773Search in Google Scholar
[23] Christiaens I, Zaragoza DB, Guilbert L, Robertson SA, Mitchell BF, Olson DM. Inflammatory processes in preterm and term parturition. J Reprod Immunol. 2008;79:50–7.10.1016/j.jri.2008.04.002Search in Google Scholar
[24] Bastek JA, Gomez LM, Elovitz MA. The role of inflammation and infection in preterm birth. Clin Perinatol. 2011;38:385–406.10.1016/j.clp.2011.06.003Search in Google Scholar
[25] Agrawal V, Hirsch E. Intrauterine infection and preterm labor. Semin Fetal Neonatal Med. 2012;17:12–9.10.1016/j.siny.2011.09.001Search in Google Scholar
[26] Kemp MW. Preterm birth, intrauterine infection, and fetal inflammation. Front Immunol. 2014;5:574.10.3389/fimmu.2014.00574Search in Google Scholar
[27] Romero R, Chaemsaithong P, Korzeniewski SJ, Tarca AL, Bhatti G, Xu Z, et al. Clinical chorioamnionitis at term II: the intra-amniotic inflammatory response. J Perinat Med. 2016;44:5–22.10.1515/jpm-2015-0045Search in Google Scholar
[28] Gomez R, Romero R, Ghezzi F, Yoon BH, Mazor M, Berry SM. The fetal inflammatory response syndrome. Am J Obstet Gynecol. 1998;179:194–202.10.1016/S0002-9378(98)70272-8Search in Google Scholar
[29] Romero R, Chaiworapongsa T, Espinoza J. Micronutrients and intrauterine infection, preterm birth and the fetal inflammatory response syndrome. J Nutr. 2003;133(5 Suppl 2):1668S–73S.10.1093/jn/133.5.1668SSearch in Google Scholar PubMed
[30] Gotsch F, Romero R, Kusanovic JP, Mazaki-Tovi S, Pineles BL, Erez O, et al. The fetal inflammatory response syndrome. Clin Obstet Gynecol. 2007;50:652–83.10.1097/GRF.0b013e31811ebef6Search in Google Scholar PubMed
[31] Romero R, Chaemsaithong P, Docheva N, Korzeniewski SJ, Tarca AL, Bhatti G, et al. Clinical chorioamnionitis at term V: umbilical cord plasma cytokine profile in the context of a systemic maternal inflammatory response. J Perinat Med. 2016;44:53–76.10.1515/jpm-2015-0121Search in Google Scholar PubMed PubMed Central
[32] Romero R, Chaemsaithong P, Docheva N, Korzeniewski SJ, Tarca AL, Bhatti G, et al. Clinical chorioamnionitis at term IV: the maternal plasma cytokine profile. J Perinat Med. 2016;44:77–98.10.1515/jpm-2015-0103Search in Google Scholar
[33] Romero R, Quintero R, Nores J, Avila C, Mazor M, Hanaoka S, et al. Amniotic fluid white blood cell count: a rapid and simple test to diagnose microbial invasion of the amniotic cavity and predict preterm delivery. Am J Obstet Gynecol. 1991;165(4 Pt 1):821–30.10.1016/0002-9378(91)90423-OSearch in Google Scholar
[34] Romero R, Yoon BH, Mazor M, Gomez R, Diamond MP, Kenney JS, et al. The diagnostic and prognostic value of amniotic fluid white blood cell count, glucose, interleukin-6, and gram stain in patients with preterm labor and intact membranes. Am J Obstet Gynecol. 1993;169:805–16.10.1016/0002-9378(93)90009-8Search in Google Scholar
[35] Romero R, Yoon BH, Mazor M, Gomez R, Gonzalez R, Diamond MP, et al. A comparative study of the diagnostic performance of amniotic fluid glucose, white blood cell count, interleukin-6, and gram stain in the detection of microbial invasion in patients with preterm premature rupture of membranes. Am J Obstet Gynecol. 1993;169:839–51.10.1016/0002-9378(93)90014-ASearch in Google Scholar
[36] Gomez R, Romero R, Galasso M, Behnke E, Insunza A, Cotton DB. The value of amniotic fluid interleukin-6, white blood cell count, and gram stain in the diagnosis of microbial invasion of the amniotic cavity in patients at term. Am J Reprod Immunol. 1994;32:200–10.10.1111/j.1600-0897.1994.tb01115.xSearch in Google Scholar
[37] Yoon BH, Yang SH, Jun JK, Park KH, Kim CJ, Romero R. Maternal blood C-reactive protein, white blood cell count, and temperature in preterm labor: a comparison with amniotic fluid white blood cell count. Obstet Gynecol. 1996;87:231–7.10.1016/0029-7844(95)00380-0Search in Google Scholar
[38] Romero R, Brody DT, Oyarzun E, Mazor M, Wu YK, Hobbins JC, et al. Infection and labor. III. Interleukin-1: a signal for the onset of parturition. Am J Obstet Gynecol. 1989;160(5 Pt 1): 1117–23.10.1016/0020-7292(90)90214-6Search in Google Scholar
[39] Romero R, Mazor M, Brandt F, Sepulveda W, Avila C, Cotton DB, et al. Interleukin-1 alpha and interleukin-1 beta in preterm and term human parturition. Am J Reprod Immunol. 1992;27:117–23.10.1111/j.1600-0897.1992.tb00737.xSearch in Google Scholar PubMed
[40] Hillier SL, Witkin SS, Krohn MA, Watts DH, Kiviat NB, Eschenbach DA. The relationship of amniotic fluid cytokines and preterm delivery, amniotic fluid infection, histologic chorioamnionitis, and chorioamnion infection. Obstet Gynecol. 1993;81:941–8.Search in Google Scholar
[41] Figueroa R, Garry D, Elimian A, Patel K, Sehgal PB, Tejani N. Evaluation of amniotic fluid cytokines in preterm labor and intact membranes. J Matern Fetal Neonatal Med. 2005;18: 241–7.10.1080/13506120500223241Search in Google Scholar PubMed
[42] Marconi C, de Andrade Ramos BR, Peracoli JC, Donders GG, da Silva MG. Amniotic fluid interleukin-1 beta and interleukin-6, but not interleukin-8 correlate with microbial invasion of the amniotic cavity in preterm labor. Am J Reprod Immunol. 2011;65:549–56.10.1111/j.1600-0897.2010.00940.xSearch in Google Scholar PubMed
[43] Romero R, Avila C, Santhanam U, Sehgal PB. Amniotic fluid interleukin 6 in preterm labor. Association with infection. J Clin Invest. 1990;85:1392–400.10.1172/JCI114583Search in Google Scholar
[44] Santhanam U, Avila C, Romero R, Viguet H, Ida N, Sakurai S, et al. Cytokines in normal and abnormal parturition: elevated amniotic fluid interleukin-6 levels in women with premature rupture of membranes associated with intrauterine infection. Cytokine. 1991;3:155–63.10.1016/1043-4666(91)90037-ESearch in Google Scholar
[45] Romero R, Sepulveda W, Kenney JS, Archer LE, Allison AC, Sehgal PB. Interleukin 6 determination in the detection of microbial invasion of the amniotic cavity. Ciba Found Symp. 1992;167:205–20; discussion 20–3.10.1002/9780470514269.ch13Search in Google Scholar
[46] Romero R, Yoon BH, Kenney JS, Gomez R, Allison AC, Sehgal PB. Amniotic fluid interleukin-6 determinations are of diagnostic and prognostic value in preterm labor. Am J Reprod Immunol. 1993;30:167–83.10.1111/j.1600-0897.1993.tb00618.xSearch in Google Scholar
[47] Saito S, Kasahara T, Kato Y, Ishihara Y, Ichijo M. Elevation of amniotic fluid interleukin 6 (IL-6), IL-8 and granulocyte colony stimulating factor (G-CSF) in term and preterm parturition. Cytokine. 1993;5:81–8.10.1016/1043-4666(93)90027-3Search in Google Scholar
[48] Yoon BH, Romero R, Kim CJ, Jun JK, Gomez R, Choi JH, et al. Amniotic fluid interleukin-6: a sensitive test for antenatal diagnosis of acute inflammatory lesions of preterm placenta and prediction of perinatal morbidity. Am J Obstet Gynecol. 1995;172:960–70.10.1016/0002-9378(95)90028-4Search in Google Scholar
[49] Hsu CD, Meaddough E, Aversa K, Hong SF, Lu LC, Jones DC, et al. Elevated amniotic fluid levels of leukemia inhibitory factor, interleukin 6, and interleukin 8 in intra-amniotic infection. Am J Obstet Gynecol. 1998;179:1267–70.10.1016/S0002-9378(98)70144-9Search in Google Scholar
[50] Gonzalez-Bosquet E, Cerqueira MJ, Dominguez C, Gasser I, Bermejo B, Cabero L. Amniotic fluid glucose and cytokines values in the early diagnosis of amniotic infection in patients with preterm labor and intact membranes. J Matern Fetal Med. 1999;8:155–8.10.3109/14767059909020480Search in Google Scholar
[51] Yoon BH, Romero R, Moon JB, Shim SS, Kim M, Kim G, et al. Clinical significance of intra-amniotic inflammation in patients with preterm labor and intact membranes. Am J Obstet Gynecol. 2001;185:1130–6.10.1067/mob.2001.117680Search in Google Scholar PubMed
[52] Jacobsson B, Mattsby-Baltzer I, Hagberg H. Interleukin-6 and interleukin-8 in cervical and amniotic fluid: relationship to microbial invasion of the chorioamniotic membranes. Br J Obstet Gynaecol. 2005;112:719–24.10.1111/j.1471-0528.2005.00536.xSearch in Google Scholar PubMed
[53] Holst RM, Laurini R, Jacobsson B, Samuelsson E, Savman K, Doverhag C, et al. Expression of cytokines and chemokines in cervical and amniotic fluid: relationship to histological chorioamnionitis. J Matern Fetal Neonatal Med. 2007;20:885–93.10.1080/14767050701752601Search in Google Scholar
[54] Combs CA, Gravett M, Garite TJ, Hickok DE, Lapidus J, Porreco R, et al. Amniotic fluid infection, inflammation, and colonization in preterm labor with intact membranes. Am J Obstet Gynecol. 2014;210:125.e1–5.10.1016/j.ajog.2013.11.032Search in Google Scholar
[55] Cobo T, Jacobsson B, Kacerovsky M, Hougaard DM, Skogstrand K, Gratacos E, et al. Systemic and local inflammatory response in women with preterm prelabor rupture of membranes. PLoS One. 2014;9:e85277.10.1371/journal.pone.0085277Search in Google Scholar
[56] Romero R, Manogue KR, Mitchell MD, Wu YK, Oyarzun E, Hobbins JC, et al. Infection and labor. IV. Cachectin-tumor necrosis factor in the amniotic fluid of women with intraamniotic infection and preterm labor. Am J Obstet Gynecol. 1989;161:336–41.10.1016/0020-7292(90)91071-WSearch in Google Scholar
[57] Romero R, Mazor M, Sepulveda W, Avila C, Copeland D, Williams J. Tumor necrosis factor in preterm and term labor. Am J Obstet Gynecol. 1992;166:1576–87.10.1016/0002-9378(92)91636-OSearch in Google Scholar
[58] Arntzen KJ, Kjollesdal AM, Halgunset J, Vatten L, Austgulen R. TNF, IL-1, IL-6, IL-8 and soluble TNF receptors in relation to chorioamnionitis and premature labor. J Perinat Med. 1998;26:17–26.10.1515/jpme.1998.26.1.17Search in Google Scholar
[59] Romero R, Ceska M, Avila C, Mazor M, Behnke E, Lindley I. Neutrophil attractant/activating peptide-1/interleukin-8 in term and preterm parturition. Am J Obstet Gynecol. 1991; 165(4 Pt 1):813–20.10.1016/0002-9378(91)90422-NSearch in Google Scholar
[60] Cherouny PH, Pankuch GA, Romero R, Botti JJ, Kuhn DC, Demers LM, et al. Neutrophil attractant/activating peptide-1/interleukin-8: association with histologic chorioamnionitis, preterm delivery, and bioactive amniotic fluid leukoattractants. Am J Obstet Gynecol. 1993;169:1299–303.10.1016/0002-9378(93)90297-VSearch in Google Scholar
[61] Puchner T, Egarter C, Wimmer C, Lederhilger F, Weichselbraun I. Amniotic fluid interleukin-8 as a marker for intraamniotic infection. Arch Gynecol Obstet. 1993;253:9–14.10.1007/BF02770627Search in Google Scholar PubMed
[62] Witt A, Berger A, Gruber CJ, Petricevic L, Apfalter P, Husslein P. IL-8 concentrations in maternal serum, amniotic fluid and cord blood in relation to different pathogens within the amniotic cavity. J Perinat Med. 2005;33:22–6.10.1515/JPM.2005.003Search in Google Scholar PubMed
[63] Mittal P, Romero R, Kusanovic JP, Edwin SS, Gotsch F, Mazaki-Tovi S, et al. CXCL6 (granulocyte chemotactic protein-2): a novel chemokine involved in the innate immune response of the amniotic cavity. Am J Reprod Immunol. 2008;60:246–57.10.1016/j.ajog.2007.10.215Search in Google Scholar
[64] Romero R, Gomez R, Galasso M, Munoz H, Acosta L, Yoon BH, et al. Macrophage inflammatory protein-1 alpha in term and preterm parturition: effect of microbial invasion of the amniotic cavity. Am J Reprod Immunol. 1994;32:108–13.10.1111/j.1600-0897.1994.tb01101.xSearch in Google Scholar
[65] Dudley DJ, Hunter C, Mitchell MD, Varner MW. Elevations of amniotic fluid macrophage inflammatory protein-1 alpha concentrations in women during term and preterm labor. Obstet Gynecol. 1996;87:94–8.10.1016/0029-7844(95)00366-5Search in Google Scholar
[66] Cohen J, Ghezzi F, Romero R, Ghidini A, Mazor M, Tolosa JE, et al. GRO alpha in the fetomaternal and amniotic fluid compartments during pregnancy and parturition. Am J Reprod Immunol. 1996;35:23–9.10.1111/j.1600-0897.1996.tb00004.xSearch in Google Scholar
[67] Hsu CD, Meaddough E, Aversa K, Copel JA. The role of amniotic fluid L-selectin, GRO-alpha, and interleukin-8 in the pathogenesis of intraamniotic infection. Am J Obstet Gynecol. 1998;178:428–32.10.1016/S0002-9378(98)70414-4Search in Google Scholar
[68] Keelan JA, Yang J, Romero RJ, Chaiworapongsa T, Marvin KW, Sato TA, et al. Epithelial cell-derived neutrophil-activating peptide-78 is present in fetal membranes and amniotic fluid at increased concentrations with intra-amniotic infection and preterm delivery. Biol Reprod. 2004;70:253–9.10.1095/biolreprod.103.016204Search in Google Scholar
[69] Jacobsson B, Holst RM, Wennerholm UB, Andersson B, Lilja H, Hagberg H. Monocyte chemotactic protein-1 in cervical and amniotic fluid: relationship to microbial invasion of the amniotic cavity, intra-amniotic inflammation, and preterm delivery. Am J Obstet Gynecol. 2003;189:1161–7.10.1067/S0002-9378(03)00594-5Search in Google Scholar
[70] Esplin MS, Romero R, Chaiworapongsa T, Kim YM, Edwin S, Gomez R, et al. Monocyte chemotactic protein-1 is increased in the amniotic fluid of women who deliver preterm in the presence or absence of intra-amniotic infection. J Matern Fetal Neonatal Med. 2005;17:365–73.10.1080/14767050500141329Search in Google Scholar
[71] Jacobsson B, Holst RM, Andersson B, Hagberg H. Monocyte chemotactic protein-2 and -3 in amniotic fluid: relationship to microbial invasion of the amniotic cavity, intra-amniotic inflammation and preterm delivery. Acta Obstet Gynecol Scand. 2005;84:566–71.10.1067/S0002-9378(03)00594-5Search in Google Scholar
[72] Athayde N, Romero R, Maymon E, Gomez R, Pacora P, Yoon BH, et al. Interleukin 16 in pregnancy, parturition, rupture of fetal membranes, and microbial invasion of the amniotic cavity. Am J Obstet Gynecol. 2000;182(1 Pt 1):135–41.10.1016/S0002-9378(00)70502-3Search in Google Scholar
[73] Pacora P, Romero R, Maymon E, Gervasi MT, Gomez R, Edwin SS, et al. Participation of the novel cytokine interleukin 18 in the host response to intra-amniotic infection. Am J Obstet Gynecol. 2000;183:1138–43.10.1067/mob.2000.108881Search in Google Scholar
[74] Chaiworapongsa T, Romero R, Espinoza J, Kim YM, Edwin S, Bujold E, et al. Macrophage migration inhibitory factor in patients with preterm parturition and microbial invasion of the amniotic cavity. J Matern Fetal Neonatal Med. 2005;18:405–16.10.1080/14767050500361703Search in Google Scholar
[75] Gotsch F, Romero R, Kusanovic JP, Erez O, Espinoza J, Kim CJ, et al. The anti-inflammatory limb of the immune response in preterm labor, intra-amniotic infection/inflammation, and spontaneous parturition at term: a role for interleukin-10. J Matern Fetal Neonatal Med. 2008;21:529–47.10.1080/14767050802127349Search in Google Scholar
[76] Romero R, Emamian M, Quintero R, Wan M, Hobbins JC, Mitchell MD. Amniotic fluid prostaglandin levels and intra-amniotic infections. Lancet. 1986;1:1380.10.1016/S0140-6736(86)91685-5Search in Google Scholar
[77] Romero R, Emamian M, Wan M, Quintero R, Hobbins JC, Mitchell MD. Prostaglandin concentrations in amniotic fluid of women with intra-amniotic infection and preterm labor. Am J Obstet Gynecol. 1987;157:1461–7.10.1016/S0002-9378(87)80245-4Search in Google Scholar
[78] Romero R, Wu YK, Mazor M, Hobbins JC, Mitchell MD. Amniotic fluid prostaglandin E2 in preterm labor. Prostaglandins Leukot Essent Fatty Acids. 1988;34:141–5.10.1016/0952-3278(88)90137-8Search in Google Scholar
[79] Romero R, Wu YK, Sirtori M, Oyarzun E, Mazor M, Hobbins JC, et al. Amniotic fluid concentrations of prostaglandin F2 alpha, 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) and 11-deoxy-13,14-dihydro-15-keto-11, 16-cyclo-prostaglandin E2 (PGEM-LL) in preterm labor. Prostaglandins. 1989;37:149–61.10.1016/0090-6980(89)90038-5Search in Google Scholar
[80] Romero R, Wu YK, Mazor M, Oyarzun E, Hobbins JC, Mitchell MD. Amniotic fluid arachidonate lipoxygenase metabolites in preterm labor. Prostaglandins Leukot Essent Fatty Acids. 1989;36:69–75.10.1016/0952-3278(89)90020-3Search in Google Scholar
[81] Bry K, Hallman M. Prostaglandins, inflammation, and preterm labor. J Perinatol. 1989;9:60–5.Search in Google Scholar
[82] Mazor M, Wiznitzer A, Maymon E, Leiberman JR, Cohen A. Changes in amniotic fluid concentrations of prostaglandins E2 and F2 alpha in women with preterm labor. Isr J Med Sci. 1990;26:425–8.Search in Google Scholar
[83] Hsu CD, Meaddough E, Aversa K, Hong SF, Lee IS, Bahodo-Singh RO, et al. Dual roles of amniotic fluid nitric oxide and prostaglandin E2 in preterm labor with intra-amniotic infection. Am J Perinatol. 1998;15:683–7.10.1055/s-2007-999302Search in Google Scholar PubMed
[84] Lee SE, Park IS, Romero R, Yoon BH. Amniotic fluid prostaglandin F2 increases even in sterile amniotic fluid and is an independent predictor of impending delivery in preterm premature rupture of membranes. J Matern Fetal Neonatal Med. 2009;22:880–6.10.1080/14767050902994648Search in Google Scholar PubMed PubMed Central
[85] Park JY, Romero R, Lee J, Chaemsaithong P, Chaiyasit N, Yoon BH. An elevated amniotic fluid prostaglandin F2α concentration is associated with intra-amniotic inflammation/infection, and clinical and histologic chorioamnionitis, as well as impending preterm delivery in patients with preterm labor and intact membranes. J Matern Fetal Neonatal Med. 2016;29:2563–72.Search in Google Scholar
[86] Fulwyler MJ. Electronic separation of biological cells by volume. Science. 1965;150:910–1.10.1126/science.150.3698.910Search in Google Scholar
[87] Wade CG, Rhyne RH, Jr., Woodruff WH, Bloch DP, Bartholomew JC. Spectra of cells in flow cytometry using a vidicon detector. J Histochem Cytochem. 1979;27:1049–52.10.1177/27.6.110874Search in Google Scholar
[88] de Graaf MT, Smitt PA, Luitwieler RL, van Velzen C, van den Broek PD, Kraan J, et al. Central memory CD4+ T cells dominate the normal cerebrospinal fluid. Cytometry B Clin Cytom. 2011;80:43–50.10.1002/cyto.b.20542Search in Google Scholar
[89] de Graaf MT, de Jongste AH, Kraan J, Boonstra JG, Sillevis Smitt PA, Gratama JW. Flow cytometric characterization of cerebrospinal fluid cells. Cytometry B Clin Cytom. 2011;80:271–81.10.1002/cyto.b.20603Search in Google Scholar
[90] Himmelfarb J, Lazarus JM, Hakim R. Reactive oxygen species production by monocytes and polymorphonuclear leukocytes during dialysis. Am J Kidney Dis. 1991;17:271–6.10.1016/S0272-6386(12)80473-2Search in Google Scholar
[91] Himmelfarb J, Hakim RM, Holbrook DG, Leeber DA, Ault KA. Detection of granulocyte reactive oxygen species formation in whole blood using flow cytometry. Cytometry. 1992;13:83–9.10.1002/cyto.990130113Search in Google Scholar PubMed
[92] Noritake M, Katsura Y, Shinomiya N, Kanatani M, Uwabe Y, Nagata N, et al. Intracellular hydrogen peroxide production by peripheral phagocytes from diabetic patients. Dissociation between polymorphonuclear leucocytes and monocytes. Clin Exp Immunol. 1992;88:269–74.10.1111/j.1365-2249.1992.tb03072.xSearch in Google Scholar PubMed PubMed Central
[93] Rabesandratana H, Dornand J. Flow cytometric analysis of oxygen species formation in activated leukocytes. Cytometry. 1993;14:695–7.10.1002/cyto.990140616Search in Google Scholar PubMed
[94] Prussin C. Cytokine flow cytometry: understanding cytokine biology at the single-cell level. J Clin Immunol. 1997;17: 195–204.10.1023/A:1027350226435Search in Google Scholar
[95] Evenson DP, Darzynkiewicz Z, Melamed MR. Simultaneous measurement by flow cytometry of sperm cell viability and mitochondrial membrane potential related to cell motility. J Histochem Cytochem. 1982;30:279–80.10.1177/30.3.6174566Search in Google Scholar PubMed
[96] Frankfurt OS. Assessment of cell viability by flow cytometric analysis using DNase exclusion. Exp Cell Res. 1983;144: 478–82.10.1016/0014-4827(83)90428-7Search in Google Scholar
[97] Xu Y, Plazyo O, Romero R, Hassan SS, Gomez-Lopez N. Isolation of Leukocytes from the Human Maternal-fetal Interface. J Vis Exp. 2015;99:e52863.10.3791/52863Search in Google Scholar
[98] McCurley TL, Larson R. Clinical applications of flow cytometry in hematology and immunology. Clin Lab Sci. 1996;9:358–62.Search in Google Scholar
[99] Delanghe JR, Kouri TT, Huber AR, Hannemann-Pohl K, Guder WG, Lun A, et al. The role of automated urine particle flow cytometry in clinical practice. Clin Chim Acta. 2000;301:1–18.10.1016/S0009-8981(00)00342-9Search in Google Scholar
[100] Savion S, Aroch I, Mammon K, Orenstein H, Fein A, Torchinsky A, et al. Effect of maternal immunopotentiation on apoptosis-associated molecules expression in teratogen-treated embryos. Am J Reprod Immunol. 2009;62:400–11.10.1111/j.1600-0897.2009.00757.xSearch in Google Scholar
[101] Arneth BM, Menschikowki M. Technology and new fluorescence flow cytometry parameters in hematological analyzers. J Clin Lab Anal. 2015;29:175–83.10.1002/jcla.21747Search in Google Scholar
[102] Bignardi GE. Flow cytometry for the microscopy of body fluids in patients with suspected infection. J Clin Pathol. 2015;68:870–8.10.1136/jclinpath-2015-203088Search in Google Scholar
[103] Xiong Y, Zhang L, Wang T. Phosphorylation of BMK1 induces prostatic carcinoma cell proliferation by promoting entry into the S phase of the cell cycle. Oncol Lett. 2016;11:99–104.10.3892/ol.2015.3909Search in Google Scholar
[104] Romero R, Mazor M, Wu YK, Sirtori M, Oyarzun E, Mitchell MD, et al. Infection in the pathogenesis of preterm labor. Semin Perinatol. 1988;12:262–79.Search in Google Scholar
[105] Romero R, Mazor M. Infection and preterm labor. Clin Obstet Gynecol. 1988;31:553–84.10.1097/00003081-198809000-00006Search in Google Scholar
[106] Romero R, Shamma F, Avila C, Jimenez C, Callahan R, Nores J, et al. Infection and labor. VI. Prevalence, microbiology, and clinical significance of intraamniotic infection in twin gestations with preterm labor. Am J Obstet Gynecol. 1990;163:757–61.10.1016/0002-9378(90)91063-ISearch in Google Scholar
[107] Romero R, Ghidini A, Mazor M, Behnke E. Microbial invasion of the amniotic cavity in premature rupture of membranes. Clin Obstet Gynecol. 1991;34:769–78.10.1097/00003081-199112000-00013Search in Google Scholar PubMed
[108] Romero R, Nores J, Mazor M, Sepulveda W, Oyarzun E, Parra M, et al. Microbial invasion of the amniotic cavity during term labor. Prevalence and clinical significance. J Reprod Med. 1993;38:543–8.Search in Google Scholar
[109] Romero R, Chaiworapongsa T, Savasan ZA, Hussein Y, Dong Z, Kusanovic JP, et al. Clinical chorioamnionitis is characterized by changes in the expression of the alarmin HMGB1 and one of its receptors, sRAGE. J Matern Fetal Neonatal Med. 2012;25:558–67.10.3109/14767058.2011.599083Search in Google Scholar
[110] Redline RW, Heller D, Keating S, Kingdom J. Placental diagnostic criteria and clinical correlation–a workshop report. Placenta. 2005;26(Suppl A):S114–7.10.1016/j.placenta.2005.02.009Search in Google Scholar
[111] Redline RW. Inflammatory responses in the placenta and umbilical cord. Semin Fetal Neonatal Med. 2006;11:296–301.10.1016/j.siny.2006.02.011Search in Google Scholar
[112] Mi Lee S, Romero R, Lee KA, Jin Yang H, Joon Oh K, Park CW, et al. The frequency and risk factors of funisitis and histologic chorioamnionitis in pregnant women at term who delivered after the spontaneous onset of labor. J Matern Fetal Neonatal Med. 2011;24:37–42.10.3109/14767058.2010.482622Search in Google Scholar
[113] Kim CJ, Romero R, Chaemsaithong P, Chaiyasit N, Yoon BH, Kim YM. Acute chorioamnionitis and funisitis: definition, pathologic features, and clinical significance. Am J Obstet Gynecol. 2015;213(4 Suppl):S29–52.10.1016/j.ajog.2015.08.040Search in Google Scholar
[114] Yoon BH, Romero R, Park JS, Kim M, Oh SY, Kim CJ, et al. The relationship among inflammatory lesions of the umbilical cord (funisitis), umbilical cord plasma interleukin 6 concentration, amniotic fluid infection, and neonatal sepsis. Am J Obstet Gynecol. 2000;183:1124–9.10.1067/mob.2000.109035Search in Google Scholar
[115] Park JS, Romero R, Yoon BH, Moon JB, Oh SY, Han SY, et al. The relationship between amniotic fluid matrix metalloproteinase-8 and funisitis. Am J Obstet Gynecol. 2001;185:1156–61.10.1067/mob.2001.117679Search in Google Scholar
[116] Pacora P, Chaiworapongsa T, Maymon E, Kim YM, Gomez R, Yoon BH, et al. Funisitis and chorionic vasculitis: the histological counterpart of the fetal inflammatory response syndrome. J Matern Fetal Neonatal Med. 2002;11:18–25.10.1080/jmf.11.1.18.25Search in Google Scholar
[117] Park CW, Lee SM, Park JS, Jun JK, Romero R, Yoon BH. The antenatal identification of funisitis with a rapid MMP-8 bedside test. J Perinat Med. 2008;36:497–502.10.1515/JPM.2008.079Search in Google Scholar
[118] Yoon BH, Romero R, Lim JH, Shim SS, Hong JS, Shim JY, et al. The clinical significance of detecting ureaplasma urealyticum by the polymerase chain reaction in the amniotic fluid of patients with preterm labor. Am J Obstet Gynecol. 2003;189:919–24.10.1067/S0002-9378(03)00839-1Search in Google Scholar
[119] DiGiulio DB, Romero R, Amogan HP, Kusanovic JP, Bik EM, Gotsch F, et al. Microbial prevalence, diversity and abundance in amniotic fluid during preterm labor: a molecular and culture-based investigation. PLoS One. 2008;3:e3056.10.1371/journal.pone.0003056Search in Google Scholar
[120] DiGiulio DB, Gervasi MT, Romero R, Vaisbuch E, Mazaki-Tovi S, Kusanovic JP, et al. Microbial invasion of the amniotic cavity in pregnancies with small-for-gestational-age fetuses. J Perinat Med. 2010;38:495–502.10.1515/jpm.2010.076Search in Google Scholar
[121] Romero R, Jimenez C, Lohda AK, et al. Amniotic fluid glucose concentration: a rapid and simple method for the detection of intraamniotic infection in preterm labor. Am J Obstet Gynecol. 1990;163:968–974.10.1016/0002-9378(90)91106-MSearch in Google Scholar
[122] Romero R, Emamian M, Quintero R, Wan M, Hobbins JC, Mazor M, et al. The value and limitations of the Gram stain examination in the diagnosis of intraamniotic infection. Am J Obstet Gynecol. 1988;159:114–9.10.1016/0002-9378(88)90503-0Search in Google Scholar
[123] Romero R, Emamian M, Quintero R, Wan M, Scioscia AL, Hobbins JC, Edberg S. Diagnosis of intra-amniotic infection: the acridine orange stain.Am J Perinatol. 1989;6:41–45.10.1055/s-2007-999542Search in Google Scholar
[124] Team RDC. A language and environment for statistical computing 2008. Available from: http://www.R-project.org.Search in Google Scholar
[125] Yoon BH, Jun JK, Park KH, Syn HC, Gomez R, Romero R. Serum C-reactive protein, white blood cell count, and amniotic fluid white blood cell count in women with preterm premature rupture of membranes. Obstet Gynecol. 1996;88:1034–40.10.1016/S0029-7844(96)00339-0Search in Google Scholar
[126] Young OM, Tang Z, Niven-Fairchild T, Tadesse S, Krikun G, Norwitz ER, et al. Toll-like receptor-mediated responses by placental Hofbauer cells (HBCs): a potential pro-inflammatory role for fetal M2 macrophages. Am J Reprod Immunol. 2015;73:22–35.10.1111/aji.12336Search in Google Scholar PubMed PubMed Central
[127] Kumar V, Sharma A. Neutrophils: Cinderella of innate immune system. Int Immunopharmacol. 2010;10:1325–34.10.1016/j.intimp.2010.08.012Search in Google Scholar PubMed
[128] Witko-Sarsat V, Rieu P, Descamps-Latscha B, Lesavre P, Halbwachs-Mecarelli L. Neutrophils: molecules, functions and pathophysiological aspects. Lab Invest. 2000;80:617–53.10.1038/labinvest.3780067Search in Google Scholar PubMed
[129] Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, et al. Neutrophil extracellular traps kill bacteria. Science. 2004;303:1532–5.10.1126/science.1092385Search in Google Scholar PubMed
[130] Stocks SC, Albrechtsen M, Kerr MA. Expression of the CD15 differentiation antigen (3-fucosyl-N-acetyl-lactosamine, LeX) on putative neutrophil adhesion molecules CR3 and NCA-160. Biochem J. 1990;268:275–80.10.1042/bj2680275Search in Google Scholar
[131] Premack BA, Schall TJ. Chemokine receptors: gateways to inflammation and infection. Nat Med. 1996;2:1174–8.10.1038/nm1196-1174Search in Google Scholar
[132] Tedder TF, Steeber DA, Pizcueta P. L-selectin-deficient mice have impaired leukocyte recruitment into inflammatory sites. J Exp Med. 1995;181:2259–64.10.1084/jem.181.6.2259Search in Google Scholar
[133] Simon SI, Burns AR, Taylor AD, Gopalan PK, Lynam EB, Sklar LA, et al. L-selectin (CD62L) cross-linking signals neutrophil adhesive functions via the Mac-1 (CD11b/CD18) beta 2-integrin. J Immunol. 1995;155:1502–14.10.4049/jimmunol.155.3.1502Search in Google Scholar
[134] Vaporciyan AA, DeLisser HM, Yan HC, Mendiguren, II, Thom SR, Jones ML, et al. Involvement of platelet-endothelial cell adhesion molecule-1 in neutrophil recruitment in vivo. Science. 1993;262:1580–2.10.1126/science.8248808Search in Google Scholar
[135] Cassatella MA. Neutrophil-derived proteins: selling cytokines by the pound. Adv Immunol. 1999;73:369–509.10.1016/S0065-2776(08)60791-9Search in Google Scholar
[136] Sampson JE, Theve RP, Blatman RN, Shipp TD, Bianchi DW, Ward BE et al. Fetal origin of amniotic fluid polymorphonuclear leukocytes. Am J Obstet Gynecol. 1997;176:77–81.10.1016/S0002-9378(97)80015-4Search in Google Scholar
[137] Lee SD, Kim MR, Hwang PG, Shim SS, Yoon BH, Kim CJ. Chorionic plate vessels as an origin of amniotic fluid neutrophils. Pathol Int. 2004;54:516–22.10.1111/j.1440-1827.2004.01659.xSearch in Google Scholar
[138] Ghezzi F, Gomez R, Romero R, Yoon BH, Edwin SS, David C, et al. Elevated interleukin-8 concentrations in amniotic fluid of mothers whose neonates subsequently develop bronchopulmonary dysplasia. Eur J Obstet Gynecol Reprod Biol. 1998;78:5–10.10.1016/S0301-2115(97)00236-4Search in Google Scholar
[139] Fortunato SJ, Menon R, Lombardi SJ. Role of tumor necrosis factor-alpha in the premature rupture of membranes and preterm labor pathways. Am J Obstet Gynecol. 2002;187:1159–62.10.1067/mob.2002.127457Search in Google Scholar PubMed
[140] Moore RM, Mansour JM, Redline RW, Mercer BM, Moore JJ. The physiology of fetal membrane rupture: insight gained from the determination of physical properties. Placenta. 2006;27:1037–51.10.1016/j.placenta.2006.01.002Search in Google Scholar PubMed
[141] Kumar D, Fung W, Moore RM, Pandey V, Fox J, Stetzer B, et al. Proinflammatory cytokines found in amniotic fluid induce collagen remodeling, apoptosis, and biophysical weakening of cultured human fetal membranes. Biol Reprod. 2006;74:29–34.10.1095/biolreprod.105.045328Search in Google Scholar
[142] Menon R, Fortunato SJ. Infection and the role of inflammation in preterm premature rupture of the membranes. Best Pract Res Clin Obstet Gynaecol. 2007;21:467–78.10.1016/j.bpobgyn.2007.01.008Search in Google Scholar
[143] Romero R, Tartakovsky B. The natural interleukin-1 receptor antagonist prevents interleukin-1-induced preterm delivery in mice. Am J Obstet Gynecol. 1992;167(4 Pt 1):1041–5.10.1016/S0002-9378(12)80035-4Search in Google Scholar
[144] Romero R, Parvizi ST, Oyarzun E, Mazor M, Wu YK, Avila C, et al. Amniotic fluid interleukin-1 in spontaneous labor at term. J Reprod Med. 1990;35:235–8.Search in Google Scholar
[145] Volkman A, Gowans JL. The origin of macrophages from bone marrow in the rat. Br J Exp Pathol. 1965;46:62–70.Search in Google Scholar
[146] Gordon S, Taylor PR. Monocyte and macrophage heterogeneity. Nat Rev Immunol. 2005;5:953–64.10.1038/nri1733Search in Google Scholar
[147] Swirski FK, Nahrendorf M, Etzrodt M, Wildgruber M, Cortez-Retamozo V, Panizzi P, et al. Identification of splenic reservoir monocytes and their deployment to inflammatory sites. Science. 2009;325:612–6.10.1126/science.1175202Search in Google Scholar
[148] Geissmann F, Jung S, Littman DR. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity. 2003;19:71–82.10.1016/S1074-7613(03)00174-2Search in Google Scholar
[149] Tacke F, Randolph GJ. Migratory fate and differentiation of blood monocyte subsets. Immunobiology. 2006;211:609–18.10.1016/j.imbio.2006.05.025Search in Google Scholar PubMed
[150] Randolph GJ, Sanchez-Schmitz G, Liebman RM, Schakel K. The CD16(+) (FcgammaRIII(+)) subset of human monocytes preferentially becomes migratory dendritic cells in a model tissue setting. J Exp Med. 2002;196:517–27.10.1084/jem.20011608Search in Google Scholar PubMed PubMed Central
[151] Passlick B, Flieger D, Ziegler-Heitbrock HW. Identification and characterization of a novel monocyte subpopulation in human peripheral blood. Blood. 1989;74:2527–34.10.1182/blood.V74.7.2527.2527Search in Google Scholar
[152] Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, Mathison JC. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science. 1990;249:1431–3.10.1126/science.1698311Search in Google Scholar PubMed
[153] Nimmerjahn F, Ravetch JV. Fc-receptors as regulators of immunity. Adv Immunol. 2007;96:179–204.10.1016/S0065-2776(07)96005-8Search in Google Scholar
[154] Ziegler-Heitbrock HW, Fingerle G, Strobel M, Schraut W, Stelter F, Schutt C, et al. The novel subset of CD14+/CD16+ blood monocytes exhibits features of tissue macrophages. Eur J Immunol. 1993;23:2053–8.10.1002/eji.1830230902Search in Google Scholar
[155] Douglas SDT, F. Chapter 67. Morphology of Monocytes and Macrophages. In: Lichtman MA, Kipps, TJ, Seligsohn U, Kaushanasky K, Prchal JT, editors. Williams Hematology, 8e. China: McGraw-Hill; 2010.Search in Google Scholar
[156] Yoon BH, Romero R, Jun JK, Park KH, Park JD, Ghezzi F, et al. Amniotic fluid cytokines (interleukin-6, tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-8) and the risk for the development of bronchopulmonary dysplasia. Am J Obstet Gynecol. 1997;177:825–30.10.1016/S0002-9378(97)70276-XSearch in Google Scholar
[157] Sadowsky DW, Adams KM, Gravett MG, Witkin SS, Novy MJ. Preterm labor is induced by intraamniotic infusions of interleukin-1beta and tumor necrosis factor-alpha but not by interleukin-6 or interleukin-8 in a nonhuman primate model. Am J Obstet Gynecol. 2006;195:1578–89.10.1016/j.ajog.2006.06.072Search in Google Scholar PubMed
The authors stated that there are no conflicts of interest regarding the publication of this article.
©2017 Walter de Gruyter GmbH, Berlin/Boston
