Objective
The objective of the study was to determine whether the bacterial load of genital mycoplasmas and gestation age are related to intraamniotic inflammatory response using the amniotic fluid levels of 18 inflammatory mediators.
Study Design
A prospective study of 145 women with singleton pregnancies complicated by preterm prelabor rupture of membranes between 24 0/7 and 36 6/7 weeks was conducted. Amniotic fluid was obtained from all women by transabdominal amniocentesis. The amounts of genital mycoplasma deoxyribonucleic acid were determined using the threshold cycle value and relative and absolute quantification techniques. A panel of multiple proteins was analyzed simultaneously using multiplex technology.
Results
Twenty-four women with the presence of genital mycoplasmas in amniotic fluid were included in the final analyses. The concentrations of 9 of the 18 evaluated proteins in the amniotic fluid correlated with bacterial load of genital mycoplasmas independent of the quantification technique used.
Conclusion
The intensity of intraamniotic inflammatory response to genital mycoplasmas decreased with gestational age.
Genital mycoplasmas ( Ureaplasma spp and Mycoplasma hominis ) belonging to the family Mycoplasmataceae and the class Mollicutes are the most common pathogens found in the amniotic fluid (AF) of pregnancies complicated by preterm prelabor rupture of membranes (PPROM). Genital mycoplasmas, commensal bacteria from the lower genital tract, are known to be difficult to cultivate because of their delicate nature. Therefore, a technique that does not involve culturing (polymerase chain reaction [PCR]) is preferred for the identification of these organisms in the AF.
The bacteria are present in the amniotic cavity of approximately 30% women who experience PPROM during pregnancy. Microorganisms inside the amniotic cavity are recognized by pattern recognition receptors, which detect the specific motifs on the surface of the microorganisms and initiate the inflammatory response cascade. The pathophysiology and clinical impact of the intraamniotic inflammatory response to bacteria or their products still have not been fully understood. Nevertheless, a strong link exists between the intraamniotic cytokine response and adverse sequelae for the mother and her preterm newborn.
The genital mycoplasmas, the smallest bacteria without a cell wall, contain lipoproteins and antigens, which following engagement with the pattern recognition receptors could initiate the host inflammatory response. Although they are historically considered to be bacteria of low virulence, their presence inside the amniotic cavity elicits a strong and robust intraamniotic inflammatory response, the intensity of which is fully comparable with other aerobic and anaerobic bacteria. Moreover, the genital mycoplasmas seem to be suitable microorganisms in which to evaluate the intraamniotic inflammatory response because they are the most common microorganism within the AF and can be easily identified using PCR.
Previous works have suggested that the bacterial load of genital mycoplasmas may be related to the intensity of the intraamniotic inflammatory response in women with PPROM or preterm labor. This hypothesis has been suggested based on either a limited number of samples or the analysis of few cytokines. Using these results as a starting point, the combination of different quantification techniques for the determination of the bacterial load of genital mycoplasmas along with the evaluation of the multiple protein panel (cytokines and neuropeptide) in AF using multiplex technologies seems to be a reliable and efficient approach to support the evidence regarding a dose-dependent intraamniotic inflammatory response to genital mycoplasmas.
Therefore, the main objective of this study was to evaluate whether the microbial burden of genital mycoplasmas is related to the intensity of the intraamniotic inflammatory response (determined by a panel of multiple proteins) and is dependent on the gestational age of PPROM pregnancies. The second aim was to compare the threshold cycle (Ct) value of the real-time PCR for genital mycoplasmas with the results of relative and absolute quantification techniques.
Materials and Methods
Sample collection
A prospective cohort study of pregnant women admitted between July 2008 and October 2010 to the Department of Obstetrics and Gynecology at the University Hospital Hradec Kralove was conducted. Inclusion criteria were the following: gestational age between 24 0/7 and 36 6/7 , singleton pregnancy, PPROM, and maternal age 18 years or older. Exclusion criteria were the following: fetal growth restriction, fetal structural malformation, the presence of chromosomal abnormalities, vaginal bleeding, signs of fetal hypoxia, and amniocentesis failure.
PPROM was defined as the rupture of fetal membranes with a leakage of AF that precedes the onset of uterine contraction by at least 2 hours and that occurs before 37 0/7 gestational weeks. This condition was diagnosed by sterile speculum examination confirming AF in the vagina in association with a positive test for the presence of insulin-like growth factor-binding protein (ACTIM PROM test; Medix Biochemica, Kauniainen, Finland) in the vaginal fluid.
Ultrasound-guided transabdominal amniocenteses were performed at admission. Approximately 5 mL of AF was aspirated before the administration of corticosteroids, antibiotics, or tocolytics. Amniocentesis is routinely offered for the assessment of the microbial status of the amniotic cavity to all women who are admitted with diagnosis of PPROM. This study was approved by the institutional review board committee (March 19, 2008; no. 200804 SO1P), and informed consent was received from all participants.
AF analyses
The sample of AF was divided into 3 polypropylene tubes and was processed immediately after sampling. The first and second tubes of uncentrifuged AF were transported to the laboratory to detect genital mycoplasmas and Chlamydia trachomatis using PCR and for aerobic and anaerobic cultivation. Protease inhibitors (Complete Mini, EDTA-free protease inhibitor cocktail; Roche Diagnostics, Basel, Switzerland) were added (40 μL per 1 mL of AF) to the third tube, which was centrifuged for 15 minutes at 2000 × g to remove cells and debris, filtered (0.22 μm using a Syringe-driven filter; TPP, Trasadingen, Switzerland), divided into aliquots, and stored at –70°C until analysis.
Detection of genital mycoplasmas
Deoxyribonucleic acid (DNA) was isolated from the AF with the QIAamp DNA minikit (QIAGEN, Hilden, Germany) according to the manufacturer’s instruction (protocol for isolation of bacterial DNA from biological fluids). Real-time PCR was performed on a Rotor-Gene 6000 instrument (QIAGEN) with the commercial kit AmpliSens C trachomatis / Ureaplasma / M hominis -FRT (Federal State Institution of Science, Central Research Institute of Epidemiology, Moscow, Russia) to detect DNA of U parvum/Urealyticum , M hominis , and C trachomatis in a common PCR tube.
The amplified product was detected using fluorescent dyes in real-time PCR. These dyes were linked to oligonucleotide probes, which bind specifically to the amplified DNA. Monitoring of the fluorescence intensities during the real-time PCR allowed for the detection of accumulating product in each detection channel. A control included PCR for beta-actin, a housekeeping gene, to examine the presence of inhibitors of the PCR.
The Ct value of the real-time PCR for genital mycoplasmas
The Ct value is the intersection between an amplification curve and a threshold line. It is a method to measure the concentration of target DNA in the PCR. Under ideal conditions (most PCR reactions are close to 100% efficient), the amount of target amplicon increases at a rate of 1 log 10 every 3.32 cycles, which means that increasing the bacterial load of genital mycoplasmas in the AF results in lower Ct values.
Relative quantification of genital mycoplasmas by PCR
The relative quantification of genital mycoplasmas in relation to the expression level of a housekeeping gene was performed using the delta Ct values. The expression level of the HLA DQ gene, which is not affected by inflammation, was measured by real-time PCR in each sample. The delta Ct value was calculated by the following formula: delta Ct value = 2 (A-B) , where A is the Ct value for genital mycoplasmas and B is the Ct value for the HLA DQ gene.
The absolute quantification of genital mycoplasmas
The amount of Ureaplasma spp DNA in copies per milliliter was determined by an absolute quantification technique using an external calibration curve. Plasmid DNA (pCR4, Invitrogen, Carlsbad, CA) was used for the preparation of the calibration curve. The concentration of Ureaplasma spp DNA in copies per microliter was converted into copies per milliliter using the following formula: concentration of Ureaplasma spp DNA (copies per microliter) × elution volume microliters)/input volume (milliliters).
Analysis of multiple proteins
Interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12, IL-17, IL-18, soluble IL-6 receptor α (sIL-6rα), adiponectin, brain-derived neurotropic factor (BDNF), C-reactive protein (CRP), granulocyte macrophage colony stimulating factor (GM-CSF), insulin-like growth factor–binding protein (IGFBP)-1, IGFBP-3, interferon-γ, leptin, monocyte chemotactic protein-1 (MCP-1), migration inhibiting factor (MIF), macrophage inflammatory protein-1α (MIP-1α), matrix metalloproteinasis-9 (MMP-9), neutropin-3 (NT-3), regulated on activation normal T-expressed and secreted (RANTES), tumor necrosis factor (TNF)-α, TNF-β, soluble TNF receptor-1 (sTNF-r1), and triggering receptor expressed on myeloid cells-1 (TREM-1) levels in AF were analyzed at the Statens Serum Institute (Department of Clinical Biochemistry and Immunology, Copenhagen, Denmark) using a multiple sandwich immunoassay based on flowmetric Luminex xMAP technology.
The samples of AF were measured undiluted in duplicate. The means of the intraassay and interassay coefficient of variation were 6% and 12%, respectively. The defined working range described by Skogstrand et al was used because of the impossibility of obtaining a cytokine-free AF sample.
The detection level was defined as half of the lowest levels in the working range for AF. Those values are as follows: IL-1β, 5 pg/mL; IL-6, 19.5 pg/mL; IL-8, 2.5 pg/mL; IL-10, 10 pg/mL; IL-12, 4 pg/mL; IL-17, 4 pg/mL; IL-18, 10 pg/mL; sIL-6rα, 19.5 pg/mL; adiponectin, 488.5 pg/mL; BDNF, 10 pg/mL; CRP, 200 pg/mL; GM-CSF, 4 pg/mL; IGFBP-1, 97.5 pg/mL; IGFBP-3, 97.5 pg/mL; interferon-γ, 4 pg/mL; leptin, 97.5 pg/mL; MCP-1, 2.5 pg/mL; MIF, 49 pg/mL; MIP-1α, 39 pg/mL; MMP-9, 244 pg/mL; NT-3, 39 pg/mL; RANTES, 2.5 pg/mL; TNF-α, 4 pg/mL; TNF-β, 4 pg/mL; sTNF-r1, 156.5 pg/mL; and TREM-1, 97.5 pg/mL. Only proteins with detectable AF levels in more than 50% of the samples were included in analyses (8 proteins were excluded from further analyses because of interferon-γ was detected in 0%; IL-12, IL-17, and TNF-α in 17%; IL-1β, IL-18, and TNF-β in 33%; and RANTES in 37%).
Statistical analysis
The demographic and clinical characteristics were compared using unpaired Student t tests for continuous variables (values are presented as the mean ± SD) or nonparametric Mann-Whitney U test (values are presented as median with the range). Categorical variables were compared using Fisher’s exact test and were presented as a count with the proportion. The normality of the data was tested using the D’Agostino and Pearson omnibus normality test and the Shapiro-Wilk test. Spearman’s rank correlation coefficient was used to analyze the correlations between the continuous variables.
Coefficients were considered statistically different from zero at the family-wise error rate of P < .05 (comparison-wise error rate α less than 0.003 regarding to Bonferroni correction). All P values were from 2-sided tests, and statistical analyses were performed using SPSS 19.0 for Mac OS X (SPSS Inc., Chicago, IL), GraphPad Prism 5.0d for Mac OS X (GraphPad Software, San Diego, CA), and PASS 11 (NCSS, Kaysville, UT).
Results
Characteristics of the study population
A total of 174 women with singleton pregnancies complicated by PPROM were admitted to the department during the study period. Twenty-nine women were excluded from the study for different reasons: fetal growth restriction (n = 11), amniocentesis failures (n = 7), age less than 18 years (n = 2), refusal to participate (n = 1), structural and/or chromosomal abnormalities (n = 3), severe vaginal bleeding (n = 3), and signs of fetal hypoxia at the time of admission (n = 2).
A total of 145 women with PPROM were included in the study. Among them, 37 (26%) had genital mycoplasmas DNA in their AF. DNA for Ureaplasma spp was detected in 35 of 37 (95%), and DNA for M hominis was detected in 2 of 37 (5%). Of the 37 women, only 24 (65%) were included in the analyses because for 7 women, there was no remaining AF in the biobank, and 6 women had polymicrobial culture (genital mycoplasmas along with aerobic or anaerobic bacteria).
The time intervals (median) between the rupture of membranes and amniocentesis were 5 hours (range, 1–23) and 4.5 hours (range, 1–22) for the whole study group and the group selected for further analyses, respectively. The difference was not significant ( P = .85). The time intervals (median) between the rupture of membranes and delivery were 35 hours (range, 5–244) and 51 hours (range, 7–120) for the whole study group and the group selected for further analyses, respectively. The difference was not significant ( P = .71). The time intervals (median) from amniocentesis to delivery were 29.5 hours (range, 3–241) and 39 hours (range, 4–114) for the whole study group and the final group, respectively. The difference was not significant ( P = .58).
Demographic data and clinical characteristics of women with the presence of genital mycoplasmas DNA in AF, as well as stratified into 2 subgroups by gestational age, are presented in Table 1 . All women self-reported to be Caucasian.
| Characteristic | The whole group 24 0/7 to 36 6/7 wks (n = 24) | Gestational age subgroup | P value a | |
|---|---|---|---|---|
| 24 0/7 to 31 6/7 wks (n = 13) | 32 0/7 to 36 6/7 wks (n = 11) | |||
| Maternal age, y | 30.1 ± 6.5 | 28.3 ± 6.8 | 31.9 ± 6.3 | .23 |
| Primiparous | 10 (42%) | 4 (31%) | 6 (82%) | .41 |
| Smoking | 10 (42%) | 7 (54%) | 3 (27%) | .24 |
| Prepregnancy BMI | 21.0 (17.0–35.7) | 20.2 (17.0–31.3) | 23.3 (17.3–35.7) | .07 |
| Gestational age at admission, d | 223 (168–251) | 215 (168–224) | 236 (226–251) | < .0001 |
| Ct value of genital mycoplasmas | 27.2 (13.2–35.0) | 23.5 (13.2–30.1) | 29.2 (19.5–35.0) | .06 |
| Ct value of HLA DQ gene | 32.1 (31.1–35.9) | 31.8 (31.1–35.9) | 32.3 (31.6–32.7) | .07 |
| Delta Ct value | 1.19 (0.92–2.37 | 1.25 (1.05–2.37) | 1.10 (0.92–1.63) | .09 |
| Amount of genital mycoplasmas DNA (copies/mL) | 3767 (13–1.77 × 10 7 ) | 9333 (320–1.77 × 10 7) | 1027 (13–8.26 × 10 5 ) | .07 |
| CRP level at admission, mg/L | 7.8 (1.0–33.0) | 9.0 (1.0–33.0) | 7.0 (1.0–21.0) | .73 |
| WBC count at admission (× 10 9 /L) | 13.0 (4.0–27.0) | 13.1 (4.0–19.0) | 11.0 (7.0–27.0) | .15 |
| Birthweight | 1708 ± 530 | 1370 ± 505 | 1940 ± 294 | .001 |
| Apgar score 5 min | 9 (0–10) | 9 (0–10) | 9 (9–10) | .06 |
| Apgar score 10 min | 10 (2–10) | 10 (2–10) | 9 (9–10) | .61 |
| The presence of HCA | 17 (71%) | 11 (85%) | 6 (55%) | .07 |
| The presence of funisitis | 8 (33) | 7 (54%) | 1 (9%) | .03 |
| Puerperal endomyometritis | 1 (4%) | 1 (8%) | 0 (0%) | 1.00 |
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