A cross-sectional study was conducted by searching our clinical database and bank of biologic samples. This study included women divided into four groups. Group 1 consisted of women in the mid-trimester (14-18 weeks) of pregnancy who underwent amniocentesis for genetic indications and delivered normal infants at term (n=84). Group 2 included women with preterm labor and intact membranes. These women were subdivided into the following categories: (a) preterm labor who delivered at term with a negative amniotic fluid culture for microorganisms (n=33), (b) preterm labor who delivered preterm (<37 weeks) with a negative amniotic fluid culture for microorganisms (n=53), and (c) preterm delivery with intra-amniotic infection (n=23). Preterm labor was defined by the presence of regular uterine contractions occurring at a frequency of at least 2 every 10 minutes and cervical changes before 37 completed weeks of gestation. Intra-amniotic infection was defined as a positive amniotic fluid culture for microorganisms and intra-amniotic inflammation as an amniotic fluid white blood cell (WBC) count of 50 cells/ml or more.[12
] Group 3 consisted of women with preterm PROM with (n=25) and without (n=26) intra-amniotic infection. PROM was diagnosed as amniorrhexis before the onset of spontaneous labor. Membrane rupture was diagnosed with the use of vaginal pooling, by ferning, or by a positive nitrazine test. The indications for amniocentesis in patients of both groups 2 and 3 were for the detection of intra-amniotic infection and fetal lung maturity. Group 4 was composed of women with term gestations (≥37 weeks of gestation) with intact membranes without intra-amniotic infection. This group was subdivided into: (a) not in labor (n=31) and (b) in labor (n=52). Women at term not in labor underwent amniocentesis for the assessment of lung maturity prior to cesarean section, whereas those in labor underwent amniocentesis because of labor at an uncertain gestational age or for the diagnosis of intra-amniotic infection. Amniotic fluid not required for clinical purposes was centrifuged at 4°C for 10 minutes and stored at −70°C. A sample of amniotic fluid was transported to the laboratory for aerobic, anaerobic, and genital Mycoplasma
cultures. Amniotic fluid WBC count and glucose concentrations were not performed in some cases. The results of these tests were used for subsequent clinical management.
Among patients with preterm labor and intact membranes who had blood sampling performed within 24 hours of amniocentesis, MIF concentrations in maternal plasma were also determined. Briefly, blood was collected into an EDTA-containing tube and centrifuged, and the supernatant was stored at −70°C. Furthermore, among patients with preterm labor with intact membranes and preterm PROM who delivered within 72 hours of amniocentesis, the presence or absence of acute inflammatory lesions in the extra-placental membranes (histologic chorioamnionitis) was assessed as previously described.[13
] This period of time was selected to preserve a meaningful temporal relationship between amniotic fluid MIF concentrations and placental pathologic findings.
All women provided informed consent prior to the collection of amniotic fluid, blood, and placental tissues. The collection of samples was approved by the Human Investigation Committees of the participating institutions and its utilization for research purposes by the IRB of the National Institute of Child Health and Human Development. Many of these samples have been used previously in studies of cytokines and arachidonic acid metabolites.
Assays for MIF in amniotic fluid and plasma
MIF concentrations in amniotic fluid and plasma were determined by using a commercially available enzyme-linked immunosorbent assay (Chemicon International, Temecula, CA). The MIF assay system was validated in our laboratory for amniotic fluid prior to determination (i.e. spike and recovery experiments). Briefly, standard and test specimens were incubated in duplicate wells of the microtiter plates coated with monoclonal antibodies against MIF. During this incubation, the immobilized antibody in the microtiter plate bound the MIF present in the standards and samples. Affinity purified monoclonal antibody to human MIF conjugated to horseradish peroxidase (HRP) was added to the wells. After an incubation period, the plate was washed to remove unbound antibody-enzyme reagents. A substrate solution (TMB: Tetramethyl Benzidine) was added and color developed in proportion to the amount of MIF bound in the initial step. Color development was stopped after a defined period and the microtiter plates were read utilizing a programmable spectrophotometer (Ceres 900 Microplate Workstation, Bio-Tek Instruments, Winooski, VT). The concentration of MIF was determined by interpolation from the respective standard curves. Inter- and intra-assay coefficients of variation (CVs) were 6.4% and 4.8%, respectively. The sensitivity was 1.4 ng/ml.
Chorioamniotic membranes were obtained from different sets of patients presenting with preterm labor and intact membranes with (n=18) and without histologic chorioamnionitis (n=20). Both groups were matched for gestational age at delivery. The membranes were dissected and washed in sterile phosphate buffered saline (PBS) (Accugene, Rockland, ME), then fixed in 10% buffered formaldehyde and embedded in paraffin. Sections of each specimen were stained with hematoxylin-eosin and examined by a pathologist (YMK) who was blinded to the study population. Five micron-thick sections of paraffin-embedded tissues were mounted on poly-L-lysine-coated microscopic glass slides (Fisher Scientific, Pittsburgh, PA). Deparaffinization, hydration, antigen retrieval, and immunostaining were performed with an automatic immunostainer (Ventana BenchMark, Ventana Medical Systems, Inc., Tucson, AZ). In brief, tissue sections were incubated at 42°C for 8 minutes with a goat polyclonal anti-human MIF antibody (R&D Systems, Minneapolis, MN) (1:100 in PBS), then 30 minutes with a biotin-conjugated rabbit anti-goat antibody (Dako, Carpinteria, CA) as a secondary antibody (1:500 in PBS), followed by the iVIEW DAB Detection kit (Ventana Medical Systems Inc.). Sections were counterstained with hematoxylin. Negative controls were obtained by replacing the specific antibody with non-immune goat immunoglobulins (R&D Systems) at the same concentration as the primary antibody. Immuno-absorption with blocking peptides of MIF (R&D Systems) were also used for negative controls.
Control absorptions were carried out under similar conditions with bovine serum albumin. At least ten microscopic fields (X400) were analyzed using a Nikon microscope with SPOT advanced software. The number of immunoreactive MIF-staining cells was computed by image analysis software (Image-ProPlus, MediaCybernetics, Silver Spring, MD). Immunoreactivity of amnion cells was semi-quantitatively analyzed by the fraction (%) of immuno-positive cells.
Real-time quantitative RT-PCR
Chorioamniotic membranes were obtained from patients with preterm labor and intact membranes with (n=13) and without histologic chorioamnionitis (n=13). Both groups were matched for gestational age at delivery. The membranes were dissected from the placentas, rinsed thoroughly with sterile ice-cold PBS (Sigma, St. Louis, MO), cut into small pieces, placed in RNAlater solution (Ambion, Austin, TX), and stored at 4°C for no longer than two weeks. Total RNA was isolated using guanidinium isothiocyanate/cesium chloride method.[14
] MIF sequences were obtained using Accession number NM_002415. Primers and a probe for real-time quantitative RT-PCR assays were designed using Primer Express software (Applied Biosystems, Foster City, CA). The primer sequences were GCAGAACCGCTCCTACAGC (forward) and TAATAGTTGATGTAGACCCTGTCCG (reverse). The probe CAGCAGGCCGCACAGCAGCT contained the fluorescent dye 6FAM at the 5′-end and TAMRA at the 3′-end. In addition, two control genes, namely the 18S ribosomal RNA and glyceraldehyde-3 phosphate dehydrogenase (GAPDH) (J04038, Applied Biosystems, Foster City, CA), were assayed with each RNA sample. The total RNA samples were treated first with RNase-free DNase (Invitrogen, Carlsbad, CA) and then used as templates to synthesize the first strand cDNA in a reaction containing Superscript II reverse transcriptase (Invitrogen, Carldbad, CA), random hexamers, and oligo-d(T) primers. Negative controls without reverse transcriptase were included in the assays. Real-time quantitative PCRs were performed using TaqMan Universal PCR Master Mix reagents and protocols in Sequence Detection System 7700 (Applied Biosystems). After PCR, amplification plots were inspected, and baselines with threshold values were set for FAM and VIC dye layers using the Sequence Detection System (SDS) software according to the manufacturer’s recommendations (Applied Biosystems). The threshold cycle numbers were computed for each well for every dye. The analysis of relative gene expression data was performed according to the method of Livak and Schmittgen.[15
Kruskal Wallis and Mann-Whitney U tests were used to determine the differences of the median amniotic fluid and plasma concentration of MIF among and between groups. Spearman rank correlation was utilized to assess the correlations. Among patients with preterm labor and intact membranes, receiver operating characteristic (ROC) curve analysis was employed for the identification of patients who had intra-amniotic infection or those who had intra-amniotic inflammation. Survival analysis and Cox proportional hazard model were applied to examine the interval from amniocentesis-to-delivery according to MIF amniotic fluid concentrations, while adjusting for confounding factors. Student’s t tests were used to examine the difference of the mean percentage of immunoreactive MIF-staining cells and the mean MIF mRNA expression in chorioamniotic membranes. A p value of <0.05 was considered statistically significant (SPSS 10.0, SPSS Inc, Chicago, Illinois).