1. INTRODUCTION
Preterm premature rupture of membranes (PPROM) has complicated 2-4% of all
pregnancies. PRROM represents, as the major identifiable cause, approximately 33%
of preterm deliveries and is strongly associated with the presence of bacteria in
amniotic cavity. Microorganisms inside amniotic cavity are recognized by the pattern
recognitions receptors. These receptors detect the specific motifs on their surface and
initiate the inflammatory response cascade1. The pathophysiology and clinical impact
of the intraamniotic inflammatory response to bacteria or their products still have not
been fully understood. Nevertheless, the strong link exists between the exhibiting of
intraamniotic cytokines responses and the adverse sequelae for mother, as well as for
preterm newborn2.
The genital mycoplasmas (U. urealyticum, U. parvum, M.
hominis), the smallest bacteria without the cell wall, contain lipoproteins and
antigens, which following engagement with pattern recognitions receptors could
initiate the host inflammatory response. Although they are historically considered as
low virulent bacteria, their presence inside the amniotic cavity elicits strong and
robust intraamniotic inflammatory response, which intensity is fully comparable with
other aerobic and anaerobic bacteria3. Moreover, the genital mycoplasmas seem to be
suitable microorganisms for the evaluation of intraamniotic inflammatory response
because of their frequent presence in amniotic fluid (AF) and the availability of PCR
identification.
2. SPECIFIC AIMS OF THE PROJECT
2.1 Aim Group 1: To evaluate the intensity of intraamniotic inflammatory response
to most common bacteria in amniotic fluid women with PPROM
2.1.1 Specific Aim A: To compare a relative [threshold cycle (Ct value)] and absolute
quantification techniques for genital mycoplasmas in AF.
2.1.2 Specific Aim B: To evaluate the relationship between the dose-dependent
intensity of intraamniotic inflammatory response to genital mycoplasmas and
gestational age.
2.1.3 Specific Aim C: To determine the association between microbial burden of
Streptococcus agalactie and intraamniotic inflammatory response.
2.2. Aim Group 2: To evaluate the intensity of intraamniotic inflammatory response
to genital mycoplasmas, Streptococcus agalactie on amnio-chorion explant model
developed from fetal membranes in different gestational ages.
2.2.1 Specific Aim D: To explore the intensity of intraamniotic inflammatory
response to genital mycoplasmas on amnio-chorion explant model.
2.2.2 Specific Aim E: To explore the relationship between the bacterial load of
Streptococcus agalactie and intraamniotic inflammatory response on amnio-chorion
explant model.
3. RESEARCH HYPOTHESIS
The recent results of our group and other have shown that intensity of intraamniotic
and intrauterine inflammation, as well as maternal systemic inflammation depend on
microbial burden of genital mycoplasmas
4-6
. Our preliminary observations suggest
that this process is age dependent. These data give rise to the hypothesis that we
would like to investigate: dose dependent intensity of intraamniotic inflammatory
response to genital mycoplasmas is probably dependent on gestational age. The
verification of our hypothesis would be consequently enable to determine the
“critical” load of genital mycoplasmas, which elicit severe intraamniotic
inflammatory response in different periods of pregnancy (24-28, 29-33, and 34-36
weeks). To determine the load, which is associated with adverse neonatal outcome.
As a next step, we would like to extend our hypothesis on the second most frequent
bacteria in AF from PPROM pregnancies - Streptococcus agalactie.
4. METHODS
4.1 The sample collection
Specific aims A, B, C: Inclusion criteria: singleton pregnancy and PPROM between
240/7 and 366/7 gestational weeks. Exclusion criteria: fetal malformation,
chromosomal abnormalities, signs of fetal hypoxia, and significant vaginal bleeding.
The transabdominal amniocentesis will be performed. Approximately 3-5 mL of AF
will be aspirated and divided into two vials. First will be sent to laboratory for PCR
analyses, second will be centrifuged; both supernatant and pellets stored at -70C until
analyses.
Specific aims D, E: Inclusion criteria: singleton pregnancy, either preterm labor
between 24-36 weeks or term pregnancy 39-41 weeks. Exclusion criteria: uterine
activity,
clinical
chorioamnionitis,
fetal
malformations,
and
chromosomal
abnormalities. Tissue appropriate for amnio-chorion explant will be collected from
placentas of women undergoing the elective caesarean section.
4.2 Quantification of DNA in AF
DNA will be isolated from the AF with the QIAamp DNA Mini Kit (QIAGEN,
Hilden, Germany). Real-time PCR will be performed on Rotor-Gene 6000 instrument
(QIAGEN, Hilden, Germany.
4.2.1 Relative quantification of genital mycoplasmas will be done by the Ct value.
4.2.2 Absolute quantification. In order to absolute quantify of amniotic fluid DNA
of these bacteria a novel real-time PCR assays will be developed. Primers and probes
targeted at the U. urealyticum, U. parvum, M. hominis, Streptococcus agalactiae
specific sequences, as well as the plasmids for these pathogens will be developed
(Generi BioTech, Hradec Kralove). The quantified plasmid standards will be
employed to establish the calibration curve for each pathogen. The calibration curves
will be used for quantification of these microorganisms in AF samples according to
the threshold cycle value for their DNA.
4.3 Cultivation of amnio-chorion explant
Dissected fetal membranes will be washed in Hank’s balanced salt solution (HBSS)
with Heparin and antibiotics and transported in this solution into the laboratory. There
will be under sterile conditions removed the maternal decidua and adherent blood
clots. Amnio-chorion will be washed three times with HBSS, and 7.5 mm circles
isolated using a biopsy punch. The tissue samples will be washed with HBSS and
placed in Faclon 12 well tissue culture plate with cultivation medium consisted of
Dulbecco’s modified Eagle’s medium/Nutrient Mixture-F12-Ham, antibiotics,
glutamine, and fetal bovine serum. The tissue sample will be inserted into 2 mL of the
cultivation medium, and cultivated at 37°C in a humified atmosphere of 5% CO2.
Media will be exchanged once in 24 hours. The stimulation will be started after 48
hours.
4.4 Preparation of bacteria for stimulation
Purchased bacterial strains (U. urealyticum, U. parvum, M. hominis, and
Streptococcus agalactie) will be cultivated in specific broth. Bacteria will be
harvested by centrifugation at 10 000 x g for 10 minutes, re-suspended in culture
media and quantified by either estimation of colony forming units (CFU) or color
changing units(CCU) for U. urealyticum, U. parvum, M. hominis, respectively
4.5 Stimulation of amnio-chorion explant
Tree different doses (102, 104, and 106 CFU/CCU) of bacterial cultures will be added
to the amnio-chorion explant model. The explant will be stimulated for 24 hours.
After that conditioned medium will be harvested and stored at -70°C until analyses.
4.6 Analyses of proteins
The AF and medium levels of selected specific proteins (cytokines, chemokines,
alarmins, and matrix metalloproteinase) will be evaluated with the employing of
enzyme-linked immunosorbent assay (ELISA) kits.
5. PROPOSED RESEARCH HARMONOGRAM
1.4.2012-31.6.2015 The recruitment women appropriate for the project.
1.4.2012-31.7.2015 Absolute quantification of genital mycoplasmas in AF.
1.1.2015-31.9.2015 Absolute quantification of Streptococcus agalactie in AF.
1.4.2012- 31.6.2015 Cultivation and stimulation of amnio-chorion explant.
1.1.2014-31.6.2015 Analysis of proteins in AF and conditioned medium.
1.7.2015-31.12.2015 The stratification data according to gestational age subgroups,
the evaluation and the analyses of data.
6. DATA REQUIREMENT AND ANALYSES
We plan to recruit at least seven women per month. A minimum sample size of 300
women in total for specific aims A, B, C provides app. 75 samples with AF genital
mycoplasmas, 15 and with Streptococcus agalactie, respectively. A minimum sample
size of 40 women (at least 10 for each gestational age subgroup) will be required for
specific aims D, E. Comparisons of nominal variables will be done with either
Fischer exact test or chi-square test. For continuous variables will be used the MannWhitney U test Kruskal-Wallis test. The Spearman correlation rank will be used for
the correlation between two independent variables. Statistical software SPSS 19 will
be used for analyses.
7. PROPOSED CO-OPERATION IN THE PROJECT
The project will be carried out in mutual cooperation among Medical Faculty in
Hradec Kralove, Faculty Hospital Hradec Kralove, University of Pardubice, Faculty
of Chemical Technology, and Hospital Pardubice.
8. DISCUSSION
The variety bacteria are presented in amniotic fluid from PPROM pregnancies. We
found the genital mycoplasmas in 25% (26/102) in our previous PPROM cohort. The
second common bacterium was Streptococcus agalactiae in 5% (5/102).
Recent studies have suggested that the intensity of inflammatory response to genital
mycoplasmas depends on microbial burden4, 5. In our previous work we proposed the
using a Ct value as a potential clinical tool for identification of bacterial load of
genital mycoplasmas in AF5. The limitation of this work was the missing validation
with absolute quantification technique, as well as a replicate cohort, which enables to
promote the Ct value as a useful clinical tool.
In order to find specific pattern of proteins, which will be valuable for the
characterisation of intraamniotic inflammatory response to bacteria, we analysed the
panel of multiple proteins (26 cytokines and neuropeptides) in 115 AF samples from
PPROM pregnancies using a multiple sandwich immunoassay based on flowmetric
Luminex xMAP technology. Our preliminary data has showed that the presence of
bacteria in AF in PPROM pregnancies is predominantly associated with higher AF
levels of L-6, IL-10, MIP-1, MMP-9, and TREM-1, but is gestational age dependent.
Moreover, we found that the bacterial load of genital mycoplasmas in AF relatively
determined by Ct value is related to the intensity of intraamniotic inflammatory
response. However, this correlation was found only below gestational age 32 weeks.
These results will be validated and explained in this proposed project in vivo and on
explant model as well.
9. THE DESCRIPTION OF PARTICIPATE FACILITIES
The members of Medical Faculty and University Hospital teams have been
systematically working on this field since 2007. The principal investigator has carried
out mutual projects with outstanding international researcher regarding preterm birth
and AF. The team of Medical Faculty is experienced with the working on amniochorion explant model, which will be a part of the project. The team of the University
Hospital Hradec Kralove will be fully engaged in the project with special focus on the
clinical area and the interpretation of the data with association to clinical outcomes.
The members of Faculty of Chemical Technology have been dealing with the clinical
isolates of human mycoplasma for more than ten years. They will be responsible for
the preparation of bacteria culture for stimulation of explant model. Members from
Hospital Pardubice have been very experienced in AF handling and the clinical
research as well. They will be responsible for AF collection.
10. EXPECTED OUTPUTS
The publications are expected in following areas: 1) the implementation of Ct value in
clinical management of women with preterm labor; 2) the comparisons between
absolute and relative quantifications techniques for AF genital mycoplasmas; 3) the
modulation of intraamniotic inflammatory response to bacteria by gestational age; 4)
the intraamniotic inflammatory response to Streptococcus agalactiae according to
microbial burden and gestational age. We expected the introduction of relative
quantification techniques (Ct value) of AF genital mycoplasmas into the clinical
management of women with PPROM below 28 weeks of gestation.
11. EXPECTED IMPACT OF THE PROJECT
The main impact of this project would be the reduction the number of preterm
newborn delivering with fetal inflammatory response syndrome, which reflects the
presence of severe intraamniotic inflammation. Therefore, we would like to determine
the “critical” bacterial loads of the most common AF bacteria, which are associated
with the severe intraamniotic inflammatory response for three different periods of
pregnancy (24-28, 29-33, and 34-37 weeks). Unique of aspect of this project will be
the validation of the clinical results on amnio-chorion explant model.
We would like to verify our preliminary results regarding relative quantification of
genital mycoplasmas by the Ct value of PCR with more precise and sophisticate
absolute quantification technique. The Ct value is routinely delivered by real-time
PCR analyses and might be a sufficient tool for the prediction of the intensity of
intraamniotic inflammation without any additional analyses (levels of cytokines).
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and
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inflammation in patients with a positive cervical fetal fibronectin. Am J Obstet
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infection with genital mycoplasmas exhibits a more intense inflammatory response
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premature rupture of membranes. Am J Obstet Gynecol 2010;203:211 e1-8. 4.
Jacobsson B, Aaltonen R, Rantakokko-Jalava K, et al. Quantification of
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