Objective
The purpose of this study was to examine the histologic and immunologic differences between fetal membrane zones after membrane rupture at term delivery.
Study Design
Fetal membrane explants from postrupture zones (periplacental, middle, rupture) were obtained from women following spontaneous vaginal delivery at term (n = 5). Tissues for histology, protein extracts, and RNA were isolated.
Results
The collagen distribution decreased and the leukocyte density increased from the periplacental zone to the rupture zone. T cells were mainly present in the rupture zone and granulocytes in the middle zone. CXCL10, CXCR1, ICAM-1, -2, PSEL, tumor necrosis factor alpha, and matrix metalloproteinase-9 levels were higher in the middle zone than in the rupture zone and periplacental zone ( P < .021). Interleukin-1beta and CXCL8 levels were higher in the rupture zone than in the middle zone and periplacental zone ( P = .018 and P < .0001).
Conclusion
During labor specific immunologic microenvironments are created in the zones of the fetal membrane that may be involved in their rupture at the end of gestation.
Normal labor begins at term with intact fetal membranes (FM) that spontaneously rupture near the end of the first stage if there is no intervention. The physiologic mechanisms that lead to FM rupture before birth are unknown although convention suggests the process is precipitated by the stress of the uterine contractions during labor. However, this fails to explain the 10% of term deliveries and 40% of preterm deliveries where membrane rupture occurs before any uterine contractions begin.
Several studies have indicated that the FM undergo a genetically programmed, biochemically mediated, maturation process near term that is characterized by collagen remodeling and apoptosis. Certain changes are limited to the rupture region of the FM overlying the cervix (termed rupture zone [RZ]). Moore et al demonstrated that the RZ is a region of physical weakness overlying the cervical opening of the uterus and is characterized by specific markers of increased collagen remodeling and apoptosis. By definition, therefore, the RZ should have different characteristics than the intact regions of the FM. It is probable that these regional characteristics develop before the onset of the contractions of labor and persist until delivery when the rupture process occurs.
In addition to the morphologic and physiologic changes of labor, an inflammatory microenvironment is created within the FM during parturition. This involves the infiltration of specific leukocyte subsets and the secretion of autocrine and paracrine primary mediators, such as proinflammatory cytokines (interleukin 1beta [IL-1β] and tumor necrosis factor alpha [TNF-α]) and chemokines (CXCL8, CXCL10). Mediators that are downstream from the immunologic include the prostaglandins and matrix metalloproteinase (MMPs), mainly MMP-9. The expression and activity of MMP-9 selectively increases during labor suggesting it has a role in the physiologic and pathologic rupture of the FM. In addition, there is increased expression of cell adhesion molecules (CAMs) that associated with “cellular spreading and homing.” These mediators may be produced and expressed by the resident cells of the reproductive tissues and/or by infiltrating leukocytes. Previously, it was demonstrated that all tissues (amnion and choriodecidua) and zones of the FM and maternal decidua contribute to the creation of an inflammatory microenvironment. The aim of this work was to identify the unique characteristics of the microenvironments in the zones of the FM, and to describe them in terms of histology, leukocyte density, and the expression of proinflammatory mediators.
Materials and Methods
Subjects and tissue collection
This study was approved by the institutional review board of the Instituto Nacional de Perinatologia Isidro Espinosa de los Reyes in Mexico City (Register 212250-02181). Written, informed consent was obtained from each subject before inclusion in the study. Subjects were excluded from the study if there was microbiologic or clinical evidence of cervicovaginal or intrauterine infection.
FM that ruptured spontaneously during labor (existence of labor: cervical dilatation ≥4 cm; contractility of the myometrium ≥3 contractions of 40 seconds within a 10 minute period by tocodynamometer) were obtained immediately after normal term (≥37 weeks) vaginal delivery from uncomplicated pregnancies. The duration of labor was similar (8-15 hours). To delimit the rupture site, we had the help of an obstetrician-gynecologist, who used a vaginal mirror to observe the cervical characteristics. If cervical dilatation was present, she stained the fetal membranes appearing in the middle of the cervical os with a gauze ball soaked in Gentian violet (Sigma-Aldrich, St. Louis, MO). This staining served as a landmark for the true rupture and was confirmed by examination of the fetal membranes after delivery. Tissues were processed within 30 minutes of delivery. In the laboratory, we used 2 long strips of the FM extending from the RZ to the placental edge and a specimen of the placental chorionic plate for our studies. Each fetal membrane zone was identified according to Figure 1 . Multiple explants of 1 cm 2 were obtained from the RZ; the middle zone (MZ), halfway between the RZ and the placental edge at least 10-12 cm from the RZ; and from the periplacental zone (PZ) comprising the 1-2 cm of thickened FM next to the edge of the placenta. FM had well-defined rupture sites and did not exhibit abnormalities, separation, or infection. Infection was indicated by the presence of massive polymorphonuclear infiltration and positive microbiologic culture. Microbiologic tests were performed in tissues by rolling a Dacron swab on the surface of the membranes. The swabs were cultured onto blood agar plates under aerobic and anaerobic conditions to ensure that tissues were free from infection. Women included in this study had internal monitoring and they were similar in ethnicity (Mexican Mestizo) and parity (primiparous). None of these women received antibiotics for prolonged rupture of fetal membranes, oxytocin augmentation, or immunosuppressive or modulating medications. Thirty-five samples were collected, however, only 5 of them were included in this study according to previously described criteria by Malak and Bell. Thirty samples were excluded because the site marked with Gentian violet did not correspond to the rupture site, suggesting that rupture did not occur spontaneously during labor. Rather, the deliveries were resolved via cesarean section or they underwent manual amniorrhexis.
The total number of explants varied between women (15-30) and depended on the length of the RZ and the distance between this zone and the placental edge. Explants were washed carefully and immediately placed in sterile saline solution to eliminate visible blood clots.
Collagen histochemistry
FM explants from each zone were fixed in 10% neutral buffered formalin (Sigma-Aldrich) for 24 hours. Tissues were dehydrated and processed for paraffin embedding. Sections (5 μM) were mounted on silane adhesive coated glass slides (Becton Dickinson, Franklin Lakes, NJ), dried overnight at room temperature, and stained with picrosirius red (0.5 g Sirius red F3B [C.I. 35782] in 500 mL saturated aqueous solution of picric acid; Sigma-Aldrich) for 1 hour. After washing in 2 changes of acidified water, slides were shaken vigorously by hand. After most of the water had been removed, sections were dehydrated in 3 changes of 100% ethanol, cleared in xylene (Sigma-Aldrich), and mounted in Entellan (Electron Microscopy Sciences, Hatfield, PA). Stained tissue sections were encoded and the blinded analysis was performed under a light microscope connected to an image capture system. Multiple fields were selected from each section.
Immunofluorescence histochemistry
Sections from each zone were blocked with 1 times phosphate buffered saline (10 mM Na 3 PO 4 , 150 mM NaCl, pH 7.2; Bio-Rad Laboratories, Hercules, CA), 1 mg mL −1 bovine serum albumin, 10 mM NaNO 3 for 30 minutes before incubation with conjugated monoclonal antibodies (1:20) for 1 hour at 37°C. We determined the phenotype of infiltrated leukocytes using anti-CD45-fluorescein isothiocyanate (FITC) (clone J33), anti-CD3-PC5 (clone UCHT1), anti-CD14-ECD (clone RM052), anti-CD19-PC7 (clone J4.119), and anti-CD56-phycoeritrin (PE) (clone N901) (I0 test; Beckman Coulter, Brea, CA). We determined the association between MMP-9 (labor mediator) and infiltrated leukocytes in these tissues by double-labeling with anti-MMP-9-FITC (clone 56129; R&D Systems, Minneapolis, MN) and anti-CD45-PE (clone J.33; I0 test; Beckman Coulter). Sections were washed in 1 times phosphate buffered saline containing 0.2% Triton (Sigma-Aldrich) (3 buffer changes, 5 minutes each) and mounted with Vectashield mounting medium (Vector Laboratories, Peterborough, UK) for visualization using the LSM 510 MetaLaser Confocal microscope (Carl Zeiss, Birmingham, UK). The histomorphometric analysis was made using encoded sections.
FM protein extracts
Immediately after delivery, FM explants from each zone were washed and special care was taken to maintain the integrity of the FM and not to separate amnion from choriodecidua; both were to be used in all experiments. Each FM explant (1 cm 2 ) was cultured in 1 mL DMEM, 1% MEM sodium pyruvate, and 1% antibiotic-antimycotic (100 U penicillin, 100 μg streptomycin, 0.25 μg amphotericin B/Ml) for 24 hours at 37°C in a humid atmosphere containing 5% CO 2 . FCS-free conditions were used in all experiments. After incubation, all explants were homogenized in their culture media using a Polytron (Brinkmann, Delran, NJ) and pooled. FM extracts from each zone were centrifuged at 14,000 × g , filtered through a 0.2 μm membrane (Corning, New York, NY), and preserved at −70° C until use.
Chemokine and cytokine concentration
Colncentrations of CXCL8, CXCL10, IL-1β, and TNF-α were measured in FM extracts from each zone using a quantitative microbead assay (Bioplex System Human Cytokine Assay; Bio-Rad Laboratories); broad range standards (1.95–32,000 pg/mL) were used. The protein content of the FM extracts was quantified by the Bradford method, and it was used to normalize the chemokine and cytokine concentrations.
Total RNA isolation and cDNA synthesis
FM explants from each zone were placed in RNAlater (Ambion, Austin, TX) to preserve RNA, and stored at −70°C until further processing. Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Total RNA was quantified by spectrophotometry and RNA integrity was verified by nondenaturing agarose gel electrophoresis. cDNA was synthesized with the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Mannheim, Germany) using random hexamer primers. The reverse transcription reaction was carried out (25°C, 10 minutes; 55°C, 30 minutes; 85°C, 5 minutes) using the Mastercycler Gradient equipment (Eppendorf, Hamburg, Germany). cDNA was stored at −20°C until use.
Real-time polymerase chain reaction (PCR)
Quantitative real-time PCR was performed using the Light Cycler 480, the Probes Master kit, and TaqMan Probes (hydrolysis probes labeled with fluorescein) according to the manufacturer’s protocol (Roche Applied Science). Specific primers for mRNA sequences of different genes were designed with the ProbeFinder software accessible at www.universalprobelibrary.com . TaqMan probes were used ( Table ). ACTB gene was the reference for normalizing purposes. To avoid false-positive signals from possible residual genomic DNA, all primers were designed to have intron spanning sequences. Five hundred nanograms of sample cDNA were added to each reaction. Real-time PCR conditions were as follows: 1 cycle at 95°C, 10 minutes, 55 cycles of denaturation (95°C, 10 seconds), annealing (60°C, 30 seconds), and extension (72°C, 1 second). Relative quantification of each molecule was calculated with the Light Cycler 480 SW 1.5 software (Roche Applied Science).
