The origin of amniotic fluid monocytes/macrophages in women with intra-amniotic inflammation or infection
-
Nardhy Gomez-Lopez
,
Roberto Romero
,
Yaozhu Leng
Abstract
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.
Introduction
Intra-amniotic inflammation/infection is a well-established etiology for spontaneous preterm labor [1], [2], [3], [4] and is strongly associated with clinical chorioamnionitis [5], [6], [7], [8], [9], [10], [11], [12], [13], [14]. This clinical condition can result from microbial invasion of the amniotic cavity, also known as microbial-induced intra-amniotic inflammation or intra-amniotic infection [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29]. Intra-amniotic inflammation can also occur in the absence of detectable microorganisms using cultivation and molecular microbiology techniques (i.e. sterile intra-amniotic inflammation) [30], [31], [32], [33], [34], a process that can be mediated by danger signals (also known as alarmins [35], [36], [37]) found in the amniotic fluid [33], [38], [39], [40], [41], [42]. Intra-amniotic inflammation/infection is characterized by an elevated number of white blood cells (WBCs) (i.e. leukocytes) in the amniotic cavity [43], [44], [45], [46], [47], [48] and increased concentrations of inflammatory mediators such as anti-microbial peptides [49], [50], [51], [52], [53], [54], cytokines [10], [33], [38], [39], [55], [56], [57], [58], [59] and lipids [60], [61], [62], [63], [64], [65], [66], [67], [68], [69], [70], [71]. Yet, the cellular immune responses in intra-amniotic infection and intra-amniotic inflammation are different [72], highlighting the importance of characterizing the specific immune cells in the amniotic cavity.
Neutrophils are the most abundant leukocyte subset in the amniotic cavity of women with inflammation/infection [43], [48]. The functions of amniotic fluid neutrophils include (1) trapping and killing pathogens in the amniotic cavity by forming web-like structures called neutrophil extracellular traps (NETs) [73], (2) phagocytizing bacteria commonly found in the lower genital tract [74] and (3) releasing antimicrobial products [29], [49], [50], [51], [52], [53], [54], [75], [76] and cytokines [48]. The origin of amniotic fluid neutrophils can be either fetal [77], [78], [79] or maternal [79], [80], or a mixture of both [79]. In the former, fetal neutrophils could derive from the chorionic plate [81] or the fetus itself (e.g. fetal lung or gastrointestinal tract) [77], [82]. In contrast, maternal neutrophils could derive from the maternal vasculature of the chorioamniotic membranes (e.g. decidual tissues) [83], [84], as has been shown for other immune cells (e.g. T cells [85]).
Following neutrophils, monocytes/macrophages are the most abundant leukocyte subset in the amniotic fluid of women with intra-amniotic inflammation/infection [48]. The classical function of monocytes is to release pro-inflammatory mediators such as cytokines [86], but these immune cells have complex functions that could vary according to the microenvironment [87], [88], [89]. We have shown that amniotic fluid monocytes/macrophages release specific cytokines in women with intra-amniotic inflammation/infection, which are different from those mediators released by neutrophils [48], indicating that these innate immune cells may display unique functions in the amniotic cavity. Early reports have suggested that monocytes/macrophages in amniotic fluid are derived from the fetus [90], [91], [92]. In line with these observations, the number of macrophages in the amniotic fluid is increased in pregnancies where fetal anomalies such as anencephaly, neural tube defects and gastroschisis are present [93], [94], [95], [96], [97], [98], [99], [100]. These findings have led to the prevailing belief that amniotic fluid monocytes/macrophages are derived from fetal tissues, particularly in the context of intra-amniotic inflammation/infection [77]. Yet, the origin of amniotic fluid monocytes/macrophages in preterm and term gestations with intra-amniotic inflammation or infection have not been fully elucidated.
The aims of this study were to (1) establish the origin of amniotic fluid monocytes/macrophages from women with intra-amniotic inflammation or infection using a highly specific technology, DNA fingerprinting, (2) assess the relationship between the origin of amniotic fluid monocytes/macrophages and preterm or term gestations and (3) associate the origin of amniotic fluid monocytes/macrophages with the localization of monocytes/macrophages in the placenta using immunohistochemistry.
Materials and methods
Study population
This was a cross-sectional study of women who underwent transabdominal amniocentesis due to clinical indications or sampling of amniotic fluid during cesarean delivery. Women were enrolled at Hutzel Women’s Hospital of the Detroit Medical Center. Amniotic fluid samples were acquired using an automatic cell counter (Cellometer Auto 2000, Nexcelom Bioscience, Lawrence, MA, USA) to obtain the viable cell numbers, most of which are WBCs or leukocytes [48]. The inclusion criteria were as follows: (1) amniotic fluid samples without blood contamination and (2) amniotic fluid samples with a large number of viable leukocytes (including mostly monocytes and neutrophils [48]) (>1×105 cells/mL) to perform fluorescence-activated cell sorting (FACS) of amniotic fluid monocytes/macrophages.
All of the women provided written informed consent to donate additional amniotic fluid for research purposes, according to protocols approved by the Institutional Review Boards of the Detroit Medical Center (Detroit, MI, USA), Wayne State University and the Perinatology Research Branch, an intramural program of the 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).
Clinical definitions
Gestational age was determined by the last menstrual period and confirmed by ultrasound examination. The gestational age derived from sonographic fetal biometry was used when 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) [7], [101], [102], [103], [104], [105]. Term delivery was defined as birth after 37 weeks of gestation, whereas preterm delivery was defined as birth between 20 and 36 6/7 weeks of gestation. Preterm deliveries were further classified as early (<28 weeks of gestation) or late (between 34 and 36 6/7 weeks of gestation) [106].
Intra-amniotic inflammation was diagnosed when the concentration of interleukin (IL)-6 in the amniotic fluid was ≥2.6 ng/mL [27], [107], [108]. Microbial invasion of the amniotic cavity was defined as a positive amniotic fluid culture [19], [20], [21], [109], [110], [111]. Intra-amniotic infection was defined as the presence of microbial invasion of the amniotic cavity with intra-amniotic inflammation [9], [27], [30], [31], [32], [33], [34], [107], [112], [113], [114], [115].
Sample collection
Amniotic fluid was either retrieved by transabdominal amniocentesis under antiseptic conditions using a 22-gauge needle monitored by ultrasound or sampled during cesarean delivery. Amniotic fluid samples were transported to the clinical laboratory in a capped sterile syringe and were cultured for aerobic and anaerobic bacteria, as well as for genital mycoplasmas [9], [116], [117], [118], [119]. Shortly after collection, a WBC count was determined in each amniotic fluid sample using a hemocytometer chamber, according to methods previously described [43]. Glucose concentration was also determined [120] and a Gram stain [121] was performed for each amniotic fluid sample. Cultures, WBC count, glucose concentration and Gram stain were not performed in all of the amniotic fluid samples collected during cesarean delivery as these samples were collected for research purposes only. However, both IL-6 concentration [48] and the presence of bacteria (bacterial live/dead staining [73], [122]) were assessed in most of the amniotic fluid samples, as previously described.
Fluorescence-activated cell sorting (FACS) of amniotic fluid monocytes/macrophages
Amniotic fluid samples were passed through a sterile 15-μm filter (Cat# 43-50015-03; pluriSelect Life Science, Leipzig, Germany) to remove epithelial cells and centrifuged at 200×g for 5 min at room temperature (n=16). The cell pellet (mostly leukocytes [73]) was washed with 1X phosphate-buffered saline (1X PBS; Life Technologies, Grand Island, NY, USA), resuspended at 1×106 cells in 100 μL of BD FACS stain buffer (Cat# 554656; BD Biosciences, San Jose, CA, USA) containing 20% human Fc receptor (FcR) blocking reagent (Cat# 130-059-901; Miltenyi Biotec, San Diego, CA, USA) and incubated for 10 min at 4°C. Next, amniotic fluid cells were incubated with the following fluorochrome-conjugated anti-human antibodies (BD Biosciences) for 30 min at 4°C in the dark: CD14-APC-Cy7 (clone MϕP9, Cat# 557831, BD Biosciences) and CD15-FITC (clone W6D3, Cat# 562370, BD Biosciences). After washing with 1X PBS, the cells were resuspended in pre-sort buffer (Cat# 563503, BD Biosciences) at a concentration of 5×106 cells/mL. Amniotic fluid monocytes/macrophages (CD14+CD15− cells) were purified using a BD FACSAria cell sorter (BD Biosciences) and BD FACSDiva 6.0 software (BD Biosciences). The purity of amniotic fluid monocytes/macrophages ranged from 71% to 98% (Figure 1). The purified monocytes/macrophages were then resuspended in RLT buffer (Qiagen, Germantown, MD, USA) and stored at −80°C until use.
Origin of amniotic fluid monocytes/macrophages.
Fluorescence-activated cell sorting (FACS) of amniotic fluid monocytes/macrophages. (A) Before cell sorting: representative flow cytometry gating strategy of an amniotic fluid sample from a woman with intra-amniotic inflammation/infection containing different cell populations: monocytes/macrophages (CD14+ cells) and neutrophils (CD15+ cells). After cell sorting: a representative image of the flow cytometry analysis of purified amniotic fluid monocytes/macrophages (CD14+CD15− cells) from a woman with intra-amniotic inflammation/infection; purity, 95.9%. (B) Origin of each sample’s amniotic fluid monocytes/macrophages based on DNA fingerprinting analysis. Red text indicates primarily fetal monocytes, blue indicates mostly maternal and gray shows a sample of mixed origin in amniotic fluid. Two pairs of samples (13&14 and 15&16) were collected from the same two patients at different gestational ages. (C) Pie chart displaying the origin of amniotic fluid monocytes/macrophages. aDetermined with AO/PI on Cellometer Auto 2000 (Nexcelom).
DNA fingerprinting
Genomic DNA was isolated from FACS-purified amniotic fluid monocytes/macrophages (n=16) using an AllPrep DNA/RNA Mini Kit (Qiagen), following the manufacturer’s instructions. Fetal or maternal genomic DNA was isolated from frozen samples of either umbilical cord or maternal blood (buffy coat) using a DNeasy Blood & Tissue Kit (Qiagen), according to the manufacturer’s instructions. DNA concentration and purity were assessed using the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). DNA samples (amniotic fluid, umbilical cord blood and maternal blood) were submitted for DNA fingerprinting to Genetica DNA Laboratories (https://www.celllineauthentication.com, Laboratory Corporation of America/LabCorp, Burlington, NC, USA). Briefly, analytical procedures for polymerase chain reaction (PCR) and capillary electrophoresis were performed on a 3130xl genetic analyzer (Applied Biosystems, Foster City, CA, USA). The 13 core CODIS STR loci plus PENTA E and PENTA D, and the gender-determining locus, amelogenin, were analyzed using the commercially available PowerPlex® 16HS amplification kit (Promega Corporation, Madison, WI, USA) and GeneMapper ID v3.2.1 software (Applied Biosystems). Appropriate positive and negative controls were concurrently used throughout the analysis. The interpretation of the DNA profile of each sample was performed by geneticists from LabCorp and returned as a written report. The reported sensitivity for this method was between 2% and 5%, indicating that this technology is capable of detecting as low as 2–5% of the DNA in question among the total DNA mixture. A cut-off of 90% was used to establish the predominant origin of amniotic fluid monocytes/macrophages.
Placental histopathological examination
Sampling of the placentas was conducted according to protocols established by the Perinatology Research Branch [123]. Five-micrometer-thick sections of formalin-fixed, paraffin-embedded tissue specimens were cut and mounted on SuperFrost Plus microscope slides (Erie Scientific LLC, Portsmouth, NH, USA). After deparaffinization, slides were rehydrated and stained with hematoxylin-eosin. A minimum of five full-thickness sections of chorionic plate, three sections of umbilical cord and three chorioamniotic membrane rolls from each case were examined by a placental pathologist who was blinded to clinical history and additional testing results. Acute inflammatory lesions of the placenta (maternal inflammatory response and fetal inflammatory response) were diagnosed according to established criteria, including staging and grading [84], [124], [125].
Immunohistochemistry
Five-micrometer-thick sections of formalin-fixed, paraffin-embedded chorioamniotic membranes, umbilical cord and chorionic plate samples from the placenta (n=12 each tissue) were cut, mounted on SuperFrost Plus microscope slides and subjected to immunochemistry using rabbit anti-human CD14 antibody (Cat# ab183322; Abcam, Cambridge, MA, USA). The detection of CD14 was used instead of that of CD68 as the former identified both monocytes and macrophages (data not shown). The staining was performed using the Leica Bond Max automatic staining system (Leica Microsystems, Wetzlar, Germany) with the Bond Polymer Refine Detection Kit (Leica Microsystems). Staining with rabbit immunoglobulin G (IgG) (Cat# ab172730; Abcam) was used as a negative control. Following staining, tissue slides were scanned using the Vectra Polaris Multispectral Imaging System (PerkinElmer, Waltham, MA, USA), and images were analyzed using the InForm 2.4.1 image analysis software (PerkinElmer).
Results
Clinical characteristics and placenta pathology of the study population
A total of 16 amniotic fluid samples were included in this study. The clinical characteristics and the placental pathology of the study population are shown in Table 1. The amniotic fluid samples had one or several of the following: (1) a positive microbiological culture, (2) an elevated concentration of IL-6 (≥2.6 ng/mL), (3) an increased WBC (>50 cells/mm3) or a viable cell (i.e. leukocytes; >100 cells/mm3) count and (4) a positive bacterial live/dead staining (Table 1). Most of the amniotic fluid samples with quantifiable glucose had a low glucose concentration (<14 mg/dL) (Table 1). Five of the samples were from women diagnosed with clinical chorioamnionitis (Table 1). Four of the amniotic fluid samples were collected from two women; for each, the first was collected during a transabdominal amniocentesis and the second was sampled during cesarean delivery (Table 1, samples 13 and 14 and samples 15 and 16). The most common microorganisms found in these amniotic fluid samples were Ureaplasma urealyticum and Mycoplasma hominis followed by Prevotella spp. (Table 1). The vast majority of women (12/14) with intra-amniotic inflammation or intra-amniotic infection presented with acute maternal and fetal inflammatory responses in the placenta (Table 1).
Clinical characteristics of amniotic fluid samples utilized for DNA fingerprinting assays.
| Sample | Predominant origin of amniotic fluid monocytes/macrophages | Clinical chorioamnionitis | Viable cell counta, cells/mm3 | Gestational age at amniocentesis, weeks | IL-6, ng/mL | Gram stain | Bacterial live/dead staining | Amniotic fluid culture | WBC, cells/mm3 | Glucose, mg/dL | Gestational age at delivery, weeks | Placental pathology results | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Acute maternal inflammatory response | Acute fetal inflammatory response | ||||||||||||
| 1 | Fetal | Yes | 786 | 22.1 | 123.7 | Negative | Negative | Negative | 390 | 7 | 23.9 | Stage 3/grade 2 | Stage 2/grade 2 |
| 2 | Fetal | No | 180 | 19 | 55.5 | Negative | Negative | Negative | 43 | 17 | 19.4 | Stage 2/grade 1 | Stage 1/grade 1 |
| 3 | Fetal | No | 286 | 39.3 | 0.5 | NA | Negative | Negative | NA | NA | 39.3 | None | None |
| 4 | Fetal | No | 508 | 23.1 | 121 | Positive | Positive | Bacteroides ureolyticus, Gardnerella vaginalis, Ureaplasma urealyticum | 750 | <1 | 23.6 | Stage 3/grade 2 | Stage 1/grade 1 |
| 5 | Fetal | No | 100 | 18.9 | 121.3 | Positive | Positive | Bacteroides fragilis | 65 | 20 | 19.6 | Stage 3/grade 2 | Stage 2/grade 1 |
| 6 | Fetal | No | 9920 | 25.7 | 27 | Positive | Positive | Enterobacter aerogenes, Enterococcus faecalis, Mycoplasma hominis, Prevotella species, Streptococcus viridans | 6938 | 4 | 25.7 | Stage 3/grade 2 | Stage 1/grade 1 |
| 7 | Fetal | No | 3660 | 21.3 | 118.7 | Negative | Positive | Staphylococcus hominis | 355 | <1 | 21.9 | Stage 3/grade 2 | Stage 2/grade 1 |
| 8 | Fetal | No | 1160 | 22.3 | 125.5 | Positive | Positive | Mycoplasma hominis, Fusobacterium nucleatum | 700 | 10 | 22.7 | Stage 2/grade 1 | Stage 1/grade 1 |
| 9 | Mix of fetal and maternal | No | 125 | 40.6 | 39.9 | NA | Positive | NA | NA | NA | 40.6 | Stage 2/grade 1 | Stage 1/grade 1 |
| 10 | Maternal | No | 9600 | 38.1 | 101.4 | Negative | Positive | Mycoplasma hominis, Ureaplasma urealyticum | NA | NA | 38.1 | Stage 2/grade 1 | Stage 2/grade 1 |
| 11 | Maternal | No | 1650 | 39.1 | 22.1 | NA | Positive | NA | NA | NA | 39.1 | Stage 1/grade 1 | Stage 1/grade 1 |
| 12 | Maternal | No | 3090 | 40.4 | 54.6 | Negative | Positive | Ureaplasma urealyticum | NA | NA | 40.4 | None | None |
| 13 | Maternal | Yes | 2200 | 35.6b | 70.6 | Positive | Positive | Mycoplasma hominis, Ureaplasma urealyticum, Prevotella spp., Streptococcus agalactiae, Streptococcus anginosus | 4000 | <1 | |||
| 14 | Maternal | Yes | 6780 | 35.6b | NA | Positive | Positive | Mycoplasma hominis, Ureaplasma urealyticum, Prevotella species | NA | NA | 35.6 | Stage 3/grade 2 | Stage 2/grade 2 |
| 15 | Fetal | Yes | 860 | 39.9c | 73.6 | Negative | Negative | Negative | 600 | <1 | |||
| 16 | Maternal | Yes | 18,800 | 40c | 47.7 | Negative | Positive | Ureaplasma urealyticum | NA | NA | 40 | Stage 2/grade 1 | Stage 2/grade 1 |
IL, interleukin; NA, not available; WBC, white blood cell. aDetermined with AO/PI on Cellometer Auto 2000 (Nexcelom). bSample 14 obtained 4 h after sample 13. cSample 16 obtained 14 h after sample 15.
Origin of amniotic fluid monocytes/macrophages
Our previous study has shown that neutrophils in the amniotic fluid can be of maternal, fetal, and/or mixed origin [79]. Herein, we sought to investigate the origin of monocytes/macrophages in the amniotic fluid of women with intra-amniotic inflammation/infection. Figure 1A shows the representative images of the flow cytometry gating strategy used to detect monocytes/macrophages in amniotic fluid from women with intra-amniotic inflammation/infection, before and after cell sorting. Amniotic fluid monocytes/macrophages were identified within the total leukocyte population by the expression of CD14 (Figure 1A). Despite neutrophils being the dominant leukocyte population in the amniotic fluid of women with intra-amniotic inflammation/infection, monocytes/macrophages were able to be isolated by cell sorting and their purity ranged from 71% to 98% (Figure 1A).
DNA fingerprinting, a highly specific technique that detects unique genetic patterns, was used to determine the origin of amniotic fluid monocytes/macrophages. As shown in Figure 1B, monocytes/macrophages of maternal and fetal origin were detected in different proportions. Samples with more than 90% of monocytes/macrophages of fetal or maternal origin were considered predominantly of that origin. An overall representation of the origin of amniotic fluid monocytes/macrophages is shown in Figure 1C.
Amniotic fluid monocytes/macrophages in women with intra-amniotic inflammation or infection can be predominantly of fetal origin
A representation of the DNA fingerprinting of amniotic fluid monocytes/macrophages of fetal origin (sample 4) is shown in Figure 2, an electropherogram containing 16 genetic sites: D3S1358, TH01, D21S11, D18S51, Penta_E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta_D, X- and Y-specific amelogenin genes, vWA, D8S1179, TPOX and FGA. The electropherogram was separated into three sections: blue, green and black. Each color indicates the dye used for the PCR multiplex: blue represents the genes amplified using fluorescein (FL dye), green represents the genes amplified using 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE dye) and black represents the genes amplified using tetramethylrhodamine (TMR dye). Each genetic site has STR alleles, which is represented by peaks. The number next to each STR allele (peak) is the number of repeats for each STR allele. Figure 1B, C shows that 56.25% (9/16) of the samples had predominant (90%–100%) fetal monocytes/macrophages in the amniotic fluid (Figure 1B, C, samples 1–8, 15; Table 1). Figure 2 illustrates that the DNA fingerprinting of amniotic fluid monocytes/macrophages is identical to the DNA fingerprinting of the fetus. By contrast, the DNA fingerprinting of amniotic fluid monocytes/macrophages differed from the one displayed by the mother (Figure 2).
DNA fingerprinting of amniotic fluid monocytes/macrophages that were mostly of fetal origin.
DNA fingerprinting of purified amniotic fluid monocytes/macrophages, the fetus (umbilical cord) and the mother (buffy coat from peripheral blood) is shown in electropherograms. Each electropherogram contains 16 genetic sites: D3S1358, TH01, D21S11, D18S51, Penta_E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta_D, X- and Y-specific amelogenin genes, vWA, D8S1179, TPOX and FGA. Each electropherogram was separated into three sections: blue, green and black. Each color indicates the dye used for the PCR multiplex: blue represents the genes amplified using fluorescein (FL dye), green represents the genes amplified using 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE dye) and black represents the genes amplified using tetramethylrhodamine (TMR dye). Each genetic site has STR alleles, which is represented by peaks. The number next to each STR allele (peak) is the number of repeats for each STR allele. The DNA fingerprinting of the amniotic fluid monocytes/macrophages is identical to the DNA fingerprinting of the fetus.
Amniotic fluid samples in which monocytes/macrophages were predominantly of fetal origin were collected from either women diagnosed with intra-amniotic infection or those with intra-amniotic inflammation without culturable microorganisms. Specifically, four of the samples with monocytes/macrophages predominantly of fetal origin (4/9) had no detectable bacteria using cultivation or microscopy techniques (Table 1). Interestingly, the majority of these samples (7/9) were collected from women who delivered early preterm neonates (<28 weeks of gestation, Table 1).
Amniotic fluid monocytes/macrophages in women with intra-amniotic infection can be predominantly of maternal origin
Figure 3 is also an electropherogram of DNA fingerprinting, which revealed that 37.5% (6/16) of the samples had mostly (96%–100%) maternal monocytes/macrophages in the amniotic fluid as shown in Figure 1B (samples 10–14, 16; Table 1). In Figure 3, the representative DNA fingerprinting of amniotic fluid monocytes/macrophages of maternal origin is shown (sample 13). The DNA fingerprinting of the amniotic fluid monocytes/macrophages is identical to the DNA fingerprinting of the mother (Figure 3). By contrast, the DNA fingerprinting of amniotic fluid monocytes/macrophages differed from the one displayed by the fetus (Figure 3).
DNA fingerprinting of amniotic fluid monocytes/macrophages that were mostly of maternal origin.
DNA fingerprinting of purified amniotic fluid monocytes/macrophages, the fetus (umbilical cord), and the mother (buffy coat from peripheral blood) is shown in electropherograms. Each electropherogram contains 16 genetic sites: D3S1358, TH01, D21S11, D18S51, Penta_E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta_D, X- and Y-specific amelogenin genes, vWA, D8S1179, TPOX and FGA. Each electropherogram was separated into three sections: blue, green and black. Each color indicates the dye used for the PCR multiplex: blue represents the genes amplified using fluorescein (FL dye), green represents the genes amplified using 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE dye) and black represents the genes amplified using tetramethylrhodamine (TMR dye). Each genetic site has STR alleles, which is represented by peaks. The number next to each STR allele (peak) is the number of repeats for each STR allele. The DNA fingerprinting of the amniotic fluid monocytes/macrophages is identical to the DNA fingerprinting of the mother.
Amniotic fluid samples in which monocytes/macrophages were predominantly of maternal origin were collected exclusively from women diagnosed with intra-amniotic infection (Table 1). None of the samples with maternal amniotic fluid monocytes/macrophages came from women with intra-amniotic inflammation without detectable microorganisms (Table 1). Strikingly, all of the women who had predominantly monocytes/macrophages of maternal origin in the amniotic fluid at the time of collection delivered term (≥37 weeks of gestation) or late preterm (34–36 completed weeks of gestation) neonates (Table 1).
A small subset of women with intra-amniotic infection can have a mixture of both fetal and maternal monocytes/macrophages in amniotic fluid
Figure 4 is also an electropherogram of DNA fingerprinting, which revealed that one woman (1/16) who was sampled during cesarean delivery had an evident mixture of fetal and maternal monocytes/macrophages in amniotic fluid as shown in Figure 1B (sample 9; Table 1). In Figure 4, the DNA fingerprinting of such amniotic fluid sample is displayed showing an evident mixture of fetal and maternal monocytes/macrophages in amniotic fluid. Therefore, the DNA fingerprinting of amniotic fluid monocytes/macrophages is a combination of the fetal and maternal STR alleles (Figure 4). The woman with a mixture of both fetal and maternal monocytes/macrophages delivered a term neonate (≥37 weeks of gestation) (Table 1).
DNA fingerprinting of amniotic fluid monocytes/macrophages that were of both fetal and maternal origin.
DNA fingerprinting of purified amniotic fluid monocytes/macrophages, the fetus (umbilical cord), and the mother (buffy coat from peripheral blood) is shown in electropherograms. Each electropherogram contains 16 genetic sites: D3S1358, TH01, D21S11, D18S51, Penta_E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta_D, X- and Y-specific amelogenin genes, vWA, D8S1179, TPOX and FGA. Each electropherogram was separated into three sections: blue, green and black. Each color indicates the dye used for the PCR multiplex: blue represents the genes amplified using fluorescein (FL dye), green represents the genes amplified using 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE dye) and black represents the genes amplified using tetramethylrhodamine (TMR dye). Each genetic site has STR alleles, which is represented by peaks. The number next to each STR allele (peak) is the number of repeats for each STR allele. The DNA fingerprinting of the amniotic fluid monocytes/macrophages included both fetal and maternal STR alleles; therefore, the DNA fingerprinting of amniotic fluid monocytes/macrophages resulted from the combination of the fetal and maternal DNAs.
Evidence that in a subset of cases the origin of amniotic fluid monocytes/macrophages may shift
One woman who was sampled twice had mostly fetal monocytes in her first amniotic fluid sample (sample 15, 860 cells/mm3, 100% of monocytes of fetal origin, Table 1 and Figure 1B). In this sample, the concentration of IL-6 was 73.6 ng/mL but bacteria were not detected by cultivation techniques or bacterial live/dead staining; therefore, this woman was diagnosed with intra-amniotic inflammation (Table 1). Fourteen hours later, a second amniotic fluid sample was collected during a cesarean delivery from the same woman (sample 16), which had a significantly larger number of leukocytes with an approximately 21-fold increase in the number of viable cells, and monocytes were mostly of maternal origin (18,800 cells/mm3, 96% of monocytes/macrophages of maternal origin, Table 1 and Figure 1B). In this second sample, the concentration of IL-6 was 47.7 ng/mL and U. urealyticum was detected by cultivation techniques and bacterial live/dead staining; therefore, this woman was diagnosed as having intra-amniotic infection (Table 1).
Another woman who underwent two amniotic fluid collections had monocytes/macrophages of exclusively maternal origin in both of the samples collected (samples 13 and 14, Figure 1B). In the first collection (sample 13, 2200 cells/mm3, Table 1) this woman presented with M. hominis, U. urealyticum, Prevotella spp., Streptococcus agalactiae and Streptococcus anginosus detected by conventional microbiological techniques and bacterial live/dead staining. She also had an elevated IL-6 concentration of 70.6 ng/mL and was thus diagnosed with intra-amniotic infection. In the second fluid collection done during cesarean delivery 4 h later, this woman still had monocytes exclusively of maternal origin (sample 14, Figure 1B) and had a higher number of leukocytes (6780 cells/mm3) with a 2-fold increase in viable cell counts. The second amniotic fluid sample cultured positive for M. hominis, U. urealyticum and Prevotella spp. as seen in the first collection, but S. agalactiae and S. anginosus were not detected this time.
Associations between the origin of amniotic fluid monocytes/macrophages and their localization in the placental tissues
Immunohistochemistry analysis revealed that monocyte/macrophage localization within the placental tissues of women with intra-amniotic inflammation/infection differs based on their origin. Figure 5A, D and G shows the varying abundance of monocytes/macrophages in the umbilical cord from women with amniotic fluid monocytes/macrophages of mostly fetal, maternal and mixed origin, respectively. The number of black arrows in each staining image corresponds to the relative quantity of CD14+ (brown) cells. A representative umbilical cord sample from a woman with amniotic fluid monocytes/macrophages of fetal origin shows many CD14+ cells spread diffusely throughout the Wharton’s jelly, suggesting a migration pattern from a vessel toward the amnion indicated by the direction of the black arrows (Figure 5A). In contrast, the presence of monocytes/macrophages in the umbilical cord of samples with maternal and mixed origin is minimal (Figure 5D, G). A magnification of these populations can be seen in the top-right corner, which clarifies the cellular patterns in each sample. Figure 5B, E and H shows the presence of monocytes/macrophages in the chorioamniotic membranes. Monocytes/macrophages in the chorioamniotic membranes were more abundant in those cases in which such cells were of maternal origin in the amniotic cavity (Figure 5E). Figure 5C, F and I shows the monocyte presence in the chorionic plate of the placenta from women with amniotic fluid monocytes/macrophages of fetal, maternal and mixed origin, respectively. Monocytes/macrophages in the chorionic plate were more abundant in those cases in which fetal cells were found in the amniotic cavity (Figure 5C) compared to the chorionic plates of both the maternal origin and mixed origin samples, which had very few monocytes/macrophages (Figure 5F, I).
Placental immunohistochemistry examination.
Immunohistochemistry staining of the umbilical cord (A, D, G), chorioamniotic membranes (B, E, H) and chorionic plate (C, F, I) obtained from women who either had mostly fetal (A–C), maternal (D–F) or a mixture of fetal and maternal (G–I) monocytes/macrophages in the amniotic fluid. CD14+ cells can be observed in brown. Magnification of monocyte/macrophage presence in the Wharton’s jelly is also shown in the top-right corner (A, D, G). Images were taken at 200× magnification. Scale bars are shown in all cases.
Discussion
Principal findings of the study
The principal findings of the study are as follows: (1) DNA fingerprinting revealed that more than 50% (56.25%, 9/16) of the amniotic fluid samples had mostly fetal monocytes/macrophages; (2) DNA fingerprinting showed that more than 30% (37.5%, 6/16) of the amniotic fluid samples had predominantly maternal monocytes/macrophages; (3) DNA fingerprinting indicated that one amniotic fluid sample (6.25%, 1/16) had a mixture of fetal and maternal monocytes/macrophages; (4) DNA fingerprinting revealed that a woman from whom two samples were analyzed (samples 15 and 16) had fetal monocytes/macrophages first, and as infection progressed, abundant maternal monocytes/macrophages invaded the amniotic cavity; (5) DNA fingerprinting revealed that another woman from whom two samples were analyzed (samples 13 and 14) had maternal monocytes/macrophages throughout the duration of infection; however, microbial burden was reduced between the first and second sample collections; (6) most samples from women who had predominantly amniotic fluid monocytes/macrophages of fetal origin delivered early preterm neonates (77.8% [7/9]); (7) all samples from women who had predominant amniotic fluid monocytes/macrophages of maternal origin delivered term or late preterm neonates (100% [6/6]); (8) the woman who had an evident mixture of fetal and maternal monocytes/macrophages in the amniotic fluid delivered a term neonate; (9) most of the women presented acute maternal and fetal inflammatory responses in the placenta (85.7% [12/14]); (10) women who had monocytes/macrophages of mostly fetal origin in amniotic fluid had abundant monocytes/macrophages (CD14+ cells) in the umbilical cord (Wharton’s jelly) and chorionic plate; and (11) women who had monocytes/macrophages of mostly maternal origin in amniotic fluid had abundant monocytes/macrophages (CD14+ cells) in the chorioamniotic membranes. Collectively, these data show that amniotic fluid monocytes/macrophages can either be predominantly of fetal or maternal origin, or a mixture of both, in women with intra-amniotic inflammation or infection.
Amniotic fluid monocytes/macrophages can be predominantly of fetal origin in women with intra-amniotic inflammation or infection
Amniotic fluid immune cells were first found to be of fetal origin in cases of intra-amniotic infection, indicating that invading microorganisms evoke a fetal response [77]. A subsequent study showed that fetal cells were predominantly present in amniotic fluid of non-human primates during non-infectious inflammation induced by IL-1β [78]. Recently, we demonstrated that the majority of amniotic fluid neutrophils are fetal in preterm gestations with intra-amniotic inflammation or infection [79]. Consistent with such previous reports, herein we found that monocytes/macrophages in the amniotic fluid of women with intra-amniotic inflammation or infection who delivered preterm neonates are mostly of fetal origin.
Cases in which amniotic fluid monocytes/macrophages were primarily fetal presented with either intra-amniotic infection, in which one or several microorganisms were detected, or intra-amniotic inflammation in the absence of detectable bacteria, a condition that in some cases may be classified as sterile intra-amniotic inflammation [30], [31], [33], [34]. However, this study did not include molecular microbiological techniques required for the diagnosis of such a clinical condition [30]. Sterile intra-amniotic inflammation has been associated with elevated concentrations of danger signals in amniotic fluid [33], [38], [39], [40], [41], [42], which can induce preterm birth in mice [126], [127]. Monocytes and resident macrophages act as sentinels through the detection of signals released by damaged or apoptotic cells [128], [129] and, therefore, are considered to be the primary cells that detect alarmins [130], [131], [132], [133]. Herein, we propose that amniotic fluid monocytes/macrophages could respond to alarmins in the setting of sterile intra-amniotic inflammation. One potential mechanism whereby alarmins induce sterile intra-amniotic inflammation is through the inflammasome [127], [134], [135], which is expressed by monocytes/macrophages [136] and serves as a platform for the caspase-1-mediated cleavage of pro-IL-1β and pro-IL-18 into their mature and bioactive forms [137], [138], [139], [140], [141], [142], [143], [144]. Inflammasome activation often results in pyroptosis, an inflammatory type of cell death first described in macrophages [145], [146], [147]. We recently showed that the effector molecule of pyroptosis, gasdermin D, is increased in amniotic fluid of women with sterile intra-amniotic inflammation who undergo preterm labor [148]. Therefore, it is likely that amniotic fluid monocytes/macrophages undergo inflammasome-mediated pyroptosis in women with sterile intra-amniotic inflammation. Alternatively, fetal macrophages may play central roles in the initiation of parturition [149], [150].
Interestingly, we observed a large number of CD14+ monocytes/macrophages in the Wharton’s jelly and chorionic plate, but few in the chorioamniotic membranes, of women whose amniotic fluid monocytes/macrophages were primarily of fetal origin. Previous studies demonstrated that concentrations of monocyte/macrophage chemoattractants such as monocyte chemoattractant protein-1 (MCP-1) are increased in amniotic fluid of women with preterm labor and intra-amniotic inflammation/infection [33], [59]. Given that the majority of women who had amniotic fluid monocytes/macrophages of fetal origin delivered early preterm neonates, it is tempting to suggest that monocytes/macrophages originating from the fetal vasculature can chemotactically migrate through the Wharton’s jelly and chorionic plate and subsequently infiltrate the amniotic cavity. Yet, further research is required to examine the mechanisms implicated in the recruitment of fetal monocytes/macrophages in this compartment, either in the setting of intra-amniotic infection or sterile intra-amniotic inflammation.
Amniotic fluid monocytes/macrophages can be predominantly of maternal origin in women with intra-amniotic infection
DNA fingerprinting revealed that amniotic fluid monocytes/macrophages can be predominantly of maternal origin in women with intra-amniotic infection. Notably, amniotic fluid monocytes/macrophages of predominantly maternal origin were only observed in samples that (1) were from late preterm or term gestations and (2) had detectable microorganisms. The genital mycoplasma U. urealyticum was found in all of these amniotic fluid samples for which a culture was performed (5/6). Further, U. urealyticum was either found alone or in culture with M. hominis, each of which has been associated with pregnancy complications including preterm labor/birth [21], [70], [109], [117], [118], [151], [152], [153], [154], [155], [156] and clinical chorioamnionitis [9], [12], [157], [158], [159], among others [160], [161], [162], [163], [164]. Genital mycoplasmas in the amniotic cavity and surrounding tissues can initiate a strong host inflammatory response, and thus, the existence of maternal monocytes/macrophages in the amniotic fluid suggests that they are present to participate in the host defense mechanisms against invading microbes as term approaches.
Maternal monocytes/macrophages could potentially migrate from the maternal vasculature of the decidua or the intervillous space and into the amniotic cavity. Indeed, previous studies suggested that neutrophils invade the amniotic cavity through this pathway [79], [80]. In line with this observation, the majority of cases in which amniotic fluid monocytes/macrophages were predominantly of maternal origin came from women whose placentas presented lesions of acute histologic chorioamnionitis (a placental lesion associated with the infiltration of neutrophils and macrophages in the chorioamniotic membranes [84], [165], [166]). Moreover, immunohistochemistry revealed that CD14+ monocytes/macrophages were abundant in the chorioamniotic membranes, but not in the umbilical cord or chorionic plate, in such cases. Together, these results suggest that in cases of intra-amniotic infection resulting in late preterm or term delivery, maternal monocytes/macrophages migrate to the amniotic cavity as part of the host response against microbes. Given that maternal peripheral monocytes are activated during spontaneous preterm labor [167], it is tempting to suggest that the activation status of such immune cells could serve as a readout of intra-amniotic inflammation/infection.
A mixture of fetal and maternal monocytes/macrophages can be found in the amniotic fluid of women with intra-amniotic infection
DNA fingerprinting showed that only one of our study patients (sample 9) had an evident mixture of maternal and fetal monocytes/macrophages in amniotic fluid. Similar to patients in which amniotic fluid monocytes/macrophages were predominantly maternal, this patient delivered at term with a high amniotic fluid IL-6 concentration and viable bacteria detected by live/dead staining, implying an intra-amniotic infection. Moreover, acute maternal and fetal inflammatory responses were observed in the placenta and umbilical cord. One possible explanation for the mixture of maternal and fetal monocytes/macrophages in amniotic fluid may be that the intra-amniotic infection was milder than in those cases at term with intra-amniotic infection, where most of the amniotic fluid monocytes/macrophages were of maternal origin.
It is worth mentioning that only one case (6.25%) included in this study had a mixture of maternal and fetal monocytes/macrophages in amniotic fluid. This is in contrast with our previous study in which we showed that a mixture of fetal and maternal neutrophils is found in 21% of cases with intra-amniotic infection/inflammation [79]. Therefore, it is likely that the immune processes regulating the migration of neutrophils and monocytes/macrophages into the amniotic cavity differ and, therefore, require further investigation.
The origin of amniotic fluid monocytes/macrophages may shift as intra-amniotic infection progresses
Two of the patients included in the current study underwent an initial transabdominal amniocentesis and were subsequently sampled again at delivery (samples 13 and 14, 15 and 16). For the first patient (samples 13 and 14), amniotic fluid monocytes/macrophages were consistently of primarily maternal origin, which may be due to the fact that both samples had a positive microbial culture. The first amniotic fluid sample obtained from the second patient (sample 15) was negative for microbiological culture and contained primarily fetal monocytes/macrophages, whereas amniotic fluid obtained at delivery (~14 h later, sample 16) was positive for U. urealyticum and now contained mostly monocytes/macrophages of maternal origin. These findings imply that the progression of intra-amniotic infection may be associated with a maternal response as indicated by the influx of maternal monocytes/macrophages into the amniotic cavity, which is a phenomenon also observed with amniotic fluid neutrophils [79].
Conclusion
In summary, monocytes/macrophages in amniotic fluid can be either predominantly of maternal or fetal origin, or a mixture of both. The detection of CD14+ monocytes/macrophages in the Wharton’s jelly and chorionic plate in cases where the majority of amniotic fluid monocytes/macrophages are of fetal origin provides a possible source for these cells, whereas maternal monocytes/macrophages may be primarily entering the amniotic cavity through the chorioamniotic membranes. Moreover, our findings suggest that the timing, severity and progression of intra-amniotic inflammation/infection may impact the maternal/fetal composition of amniotic fluid monocytes/macrophages. These findings provide evidence that both the fetus and the mother participate in the host defense mechanisms against intra-amniotic inflammation or infection.
Acknowledgments
We thank the physicians and nurses from the Center for Advanced Obstetrical Care and Research and the Intrapartum Unit for their invaluable support in collecting human samples. We also thank staff members of the PRB Clinical Laboratory for their help with processing the placental samples and Dr. Suzanne M. Jacques from the PRB Histology/Pathology Unit for the pathological examination of the histological sections. Finally, we thank Derek Miller for his critical readings of the manuscript.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: This research was supported, in part, by the Perinatology Research Branch, Division of Obstetrics and Maternal-Fetal Medicine, 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. Dr. Romero has contributed to this work as part of his official duties as an employee of the United States Federal Government. This research was also supported by the Wayne State University Perinatal Initiative in Maternal, Perinatal and Child Health.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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©2019 Walter de Gruyter GmbH, Berlin/Boston
Articles in the same Issue
- Frontmatter
- Review
- The role of complement in preterm birth and prematurity
- Original Articles – Obstetrics
- Variation in C-reactive protein at 1 month post-partum by etiology of preterm birth: selective identification of those at risk for both poor pregnancy outcome and future health complications
- Procedure related risk of premature delivery and fetal growth reduction following amniocentesis, transcervical and transabdominal chorionic villus sampling: a retrospective study
- Cervical length at 31–34 weeks of gestation: transvaginal vs. transperineal ultrasonographic approach
- The origin of amniotic fluid monocytes/macrophages in women with intra-amniotic inflammation or infection
- Placental elasticity assessment by point shear wave elastography in pregnancies with intrauterine growth restriction
- A 17-years analysis of terminations of pregnancy ≥14 weeks of gestation in a German level 1 perinatal center
- Simulation of an impacted fetal head extraction during cesarean section: description of the creation and evaluation of a new training program
- Academic tweeting in #ObGyn. Where do we stand?
- Original Articles – Fetus
- Fetal heart examination at the time of 13 weeks scan: a 5 years’ prospective study
- Comparison of fetal cardiac functions between small-for-gestational age fetuses and late-onset growth-restricted fetuses
- Original Article – Newborn
- Protocols for early discharging of premature infants: an empirical assessment on safety and savings
- Letter to the Editor
- Maternal blood pressure levels prepartum correlate with neonatal birth weight in preeclampsia
Articles in the same Issue
- Frontmatter
- Review
- The role of complement in preterm birth and prematurity
- Original Articles – Obstetrics
- Variation in C-reactive protein at 1 month post-partum by etiology of preterm birth: selective identification of those at risk for both poor pregnancy outcome and future health complications
- Procedure related risk of premature delivery and fetal growth reduction following amniocentesis, transcervical and transabdominal chorionic villus sampling: a retrospective study
- Cervical length at 31–34 weeks of gestation: transvaginal vs. transperineal ultrasonographic approach
- The origin of amniotic fluid monocytes/macrophages in women with intra-amniotic inflammation or infection
- Placental elasticity assessment by point shear wave elastography in pregnancies with intrauterine growth restriction
- A 17-years analysis of terminations of pregnancy ≥14 weeks of gestation in a German level 1 perinatal center
- Simulation of an impacted fetal head extraction during cesarean section: description of the creation and evaluation of a new training program
- Academic tweeting in #ObGyn. Where do we stand?
- Original Articles – Fetus
- Fetal heart examination at the time of 13 weeks scan: a 5 years’ prospective study
- Comparison of fetal cardiac functions between small-for-gestational age fetuses and late-onset growth-restricted fetuses
- Original Article – Newborn
- Protocols for early discharging of premature infants: an empirical assessment on safety and savings
- Letter to the Editor
- Maternal blood pressure levels prepartum correlate with neonatal birth weight in preeclampsia
