Materials and Methods
Animals and study groups
Eleven chronically catheterized pregnant pigtail macaques ( Macaca nemestrina ) at 118-125 days’ gestation (term = 172 days) received 1 of 2 experimental treatments: choriodecidual and intraamniotic saline infusions (n = 5), or intraamniotic balloon inflation with saline (n = 6; varying protocols) ( Table 1 ). In our model, pregnant pigtail macaques were time-mated and fetal age determined using early ultrasound. The tethered chronic catheter preparation was used for all in vivo experiments and is a major breakthrough in studying maternal-fetal immunologic responses. Details of our model have been described elsewhere ; we describe slight modifications necessary to create the balloon inflation model. The animal was first conditioned to a nylon jacket/tether system for several weeks before surgery. On day 118-125 of pregnancy (term = 172 days) a maternal temperature probe and catheters were implanted into the maternal femoral vein and amniotic cavity. Two or 3 intraamniotic balloons were implanted into the amniotic fluid per protocol ( Table 1 ). Care was taken to make the smallest uterine incision possible to accommodate these catheters, usually 3-4 cm in length. The saline controls did not have the intraamniotic balloons inserted, but instead had amniotic fluid, choriodecidual, maternal femoral vein and fetal vein catheters, and 1 temperature probe implanted. After surgery, the catheters were tracked through the tether system and cefazolin and terbutaline sulfate were administered to reduce postoperative infection risk and uterine activity. Terbutaline was stopped at least 72 hours before experimental start (∼5 half-lives for terbutaline, 40 half-lives for cefazolin, >97% of both drugs eliminated). Cefazolin (1 g) was administered intravenously each day to minimize chances of a catheter-related infection. Experiments began approximately 10 days to 2 weeks after catheterization surgery to allow recovery. At our center, term gestation in the noninstrumented pigtail macaque population averages 172 days. Catheterization surgery (118-125 days) and experimental start (128-135 days) are roughly equivalent to 28 and 30 weeks’ human gestation, respectively. The saline control group experiments were performed a few years before the uterine overdistention experiments and were not repeated to allow for randomization due to ethical and financial constraints. The saline control animals originally received 2 inoculations (amniotic fluid, choriodecidual) to demonstrate that neither inoculation was associated with elevated cytokines or prostaglandins.
|Experiment no.||Intraamniotic balloons||Intrapartum fetal demise||Labor within 10 d||Days to delivery|
|No.||Total volume, mL||Days to maximum inflation|
A 22-French Kenguard silicone-coated Foley catheter (Kendall Co, Mansfield, MA) designed for the human bladder was modified to create an intraamniotic balloon. First, the Foley catheter was cut close to the balloon and then a polyvinyl 1-mm catheter was glued into the Foley catheter channel to allow for balloon inflation. We determined that this Foley balloon could be inflated with 80 mL of saline and despite a variety of mechanical stresses remain inflated for >6 weeks on the bench (data not shown).
Uterine activity, labor and delivery
Intraamniotic pressure, fetal heart rate, and maternal temperature were continuously recorded and digitized with a Powerlab System (AD Instruments, Colorado Springs, CO) connected to an computer. Amniotic fluid pressure signals were processed after delivery using custom software to eliminate noise due to respiration or position changes. The area under the pressure – time curve (mm Hg × seconds, contraction area) was calculated to directly measure the strength of each contraction. To account for changes in the frequency of contractions, and to help determine how total uterine activity changes over time, we calculated the average mm Hg × seconds generated over each hour. This method of quantifying uterine activity yields numeric values with units of mm Hg × s/h, and reflects the average of the total uterine activity occurring in each hour. Labor was defined as progressive uterine activity associated with cervical effacement and dilatation (at least 1 cm dilated). For reference, the preterm macaque fetus (∼200-300 g) will deliver once the cervix is approximately 4 cm dilated. We further defined labor as associated with balloon inflation if labor occurred within 10 days of initial balloon inflation. Cervical examinations were performed on the day prior to inoculation and if labor was suspected based on uterine activity. Cesarean delivery was performed in all animals to optimize the collection of intact gestational tissues except 1 uterine stretch case that delivered spontaneously overnight. In saline controls, the cesarean delivery was performed 7 days after initial inoculation; saline was inoculated twice (day 0 and day 4) in alternating order into the choriodecidual space and amniotic fluid. At the time of cesarean delivery, the intraamniotic balloons were inspected and it was noted whether or not they were inflated. The amount of amniotic fluid at the time of catheterization surgery and cesarean delivery was recorded. Amniotic fluid was collected frequently over the course of the experiment as previously described. Fetal blood and tissues (lungs, meninges) were also cultured at the time of cesarean delivery. After cesarean delivery, fetuses were euthanized by barbiturate overdose followed by exsanguination and fetal necropsy. Complete gross and histopathologic examination was performed on infants and placentas. A board-certified veterinary pathologist (A.E.B.) examined the placenta and uterine tissues.
Quantitation of amniotic fluid cytokines and prostaglandins
Amniotic fluid and maternal and fetal blood were sampled frequently before (–24 and –1 hour) and after (+1, +6, +12, +24 hours, and then daily until delivery) balloon inflation. The amniotic fluid catheter malfunctioned in 1 animal preventing amniotic fluid sampling. Amniotic fluid and blood samples were centrifuged for 5 minutes at 1200 rpm and the supernatant frozen and stored at –80°C. Prior to freezing, ethylene diamine tetra-acetic acid (8.7 mmol/L) and indomethacin (0.3 mmol/L) was added to samples saved for cytokine and prostaglandin quantitation, respectively. Cytokine (interleukin [IL]-1β, IL-6, IL-8, and tumor necrosis factor [TNF]-α) levels were determined by Luminex multiplex technology (Austin, TX) using commercially available nonhuman primate cytokine kits (Millipore, Billerica, MA). Quantities of prostaglandin E2 (PGE 2 ) and prostaglandin F2α (PGF 2α ) were determined using commercially available human enzyme-linked immunosorbent assay kits (Cayman Chemical, Ann Arbor, MI). CCL2 levels were determined using the commercially available human enzyme-linked immunosorbent assay kit (R&D Systems Ltd, Minneapolis, MN). Values were converted to pg/mL by comparison to a standard curve.
The purpose of performing biomechanical calculations is to quantify the relative amounts of added tissue stress induced by the different protocols of the experiments. This is important, since the ability of the myometrium to adapt is likely dependent on the relationship between the volume change and the mechanical reserve of the tissue. Additionally, the baseline intrauterine pressure may be critically important in the initiation of labor.
The physiological loads on the uterus following inflation of a balloon can be divided into 3 stages. First, balloon inflation increases the intrauterine volume, which abruptly increases intrauterine pressure and tension on the wall of the uterus. From a technical perspective, the added volume increases stress on the uterine wall according to the stress-strain relationship of the tissue. Stress is the tangential force per unit area. The second stage occurs over the course of several minutes, when the tissue lengthens to accommodate the new volume through a process known as viscoelastic creep. During this stage, the intrauterine pressure and tissue stress both fall from the peak values occurring immediately after balloon inflation, but remain higher than prior to balloon inflation. Finally, over hours or days, a third stage may occur, where the myometrium adapts to the conditions by tissue remodeling. Eventually it may achieve intrauterine pressure and wall stress that are similar to the conditions before balloon inflation.
The law of Laplace can approximate the relationship between intrauterine pressure and wall stress throughout these stages. Data values for creep, strain, and stress for myometrium of pregnancy are not available for the macaque, so we approximated these values from the best available human data. Resting intrauterine pressures have previously been measured by amniocentesis in human gestation. Between 30-34 weeks, pressures measured at the abdominal surface averaged 6 torr. The mean intrauterine pressure is higher than at the abdominal surface, because there is a loss of pressure as the fluid rises (gains elevation). The distance from the abdominal surface to the center of the uterine cavity is ∼8-10 cm, so it is necessary to add the pressure corresponding to this lower height to obtain the mean intrauterine pressure in the center of the cavity. Thus, adding the pressure of 8 cm H 2 O (6 torr) to the 6 torr measured at the abdominal surface yields a typical resting intrauterine pressure of approximately 12 torr (1600 Pa). We used this value as the resting intrauterine pressure before balloon inflation in our calculations for each experiment.
To approximate the conditions through the first 2 stages following balloon inflation, the following calculations were performed:
Stage 1, balloon inflation. Before inflation, intrauterine pressure was approximated to be 12 torr. Uterine wall stress was calculated from the volume using the law of Laplace for a sphere: T = P*r/2w, where T is wall stress, P is intrauterine pressure, r is the radius of the sphere, and w is wall thickness. For the relatively small changes of r encountered in this work, changes of w were small and were ignored. After balloon inflation, the new surface area of the uterus was calculated from the new volume. The new stress was calculated from the stress-strain relationship reported for the internal os (opening) of the pregnant human cervix. A postinflation intrauterine pressure was then recalculated using the law of Laplace.
Stage 2, tissue creep. Isolated myocytes can creep to 55% of the stress values for peak stress. Since the dominant component of the wall of the uterus is the myocyte, we will use this value to approximate tissue creep. The intrauterine pressure was then recalculated using the postcreep value for stress.
An additional assumption must be made to address the anatomy of the macaque uterus. The calculations were performed using the same wall thickness (3 mm) throughout the uterus, although our measurements demonstrate that wall thickness varied between 2.9-4.2 mm in the fundal area, and is approximately 2 mm in the lower uterine segment. Balloon inflation preferentially increases wall stress over the thinner parts of the wall, and partly spares the thicker regions. However, because we are unsure of the anatomic details regarding the distribution of the wall thinning in each animal, we are unable to assess the distributions of the peak stresses. Because of this assumption, we suggest that calculated pressures be interpreted as an upper limit, and the stress increases be interpreted as a lower limit. We further refined our estimates of uterine wall stress using the weight of the fetus and amniotic fluid volume in each experiment. For unavailable data, average values of the other experiments were used. We estimated the placental volume for the preterm macaque to be 75 mL.
RNA extraction and microarray processing
To study genetic pathways in Macaca nemestrina , we used the rhesus macaque array (GeneChip rhesus macaque genome array, Affymetrix, Santa Clara, CA), which allows interrogation of 47,000 Macaca mulatta transcripts and provides comprehensive transcriptome coverage. Genetic differences between Macaca mulatta and Macaca nemestrina are predicted to be <1%, which is consistent with our published data. RNA extraction was performed using miRNeasy mini kits (Qiagen, Valencia, CA) following the manufacturer’s established protocol for purification of total RNA from animal tissues. RNA integrity was assessed with a 2100 Bioanalyzer (Agilent, Santa Clara, CA) and quantified with NanoDrop 8000 (Thermo Scientific, Waltham, MA). Processing of RNA samples was carried out according to the Affymetrix GeneChip 3’IVT express kit protocol. Briefly, these methods include the synthesis of first- and second-strand complementary DNAs (cDNAs), the purification of double-stranded cDNA, the synthesis of complementary RNA (cRNA) by in vitro transcription, the recovery and quantitation of biotin-labeled cRNA, the fragmentation of this cRNA and subsequent hybridization to the microarray slide, the posthybridization washings, and the detection of the hybridized cRNAs using a streptavidin-coupled fluorescent dye. Hybridized Affymetrix arrays were scanned with an Affymetrix GeneChip 3000 fluorescent scanner. Image generation and feature extraction was performed using Affymetrix GeneChip Command Console software.
Single gene analysis
The data discussed in this publication have been deposited in National Center for Biotechnology Information’s Gene Expression Omnibus (GEO; www.ncbi.nlm.nih.gov/geo/index.cgi ) and are accessible through GEO series accession number GSE63274. Analysis of the microarray data focused first on differential expression of single genes. Raw microarray data were preprocessed and analyzed with the Bioconductor ( www.bioconductor.org/ ) oligo package. Several quality control steps were carried out to insure that the data were of high quality: (1) visual inspection of the chip pseudoimages generated by the Bioconductor affyPLM package ; (2) generation and inspection of histograms of raw signal intensities; (3) principal components analysis plots of the normalized data; and (4) generation and comparison of the relative log expression and normalized unscaled SE using the Bioconductor affyPLM package. The data were normalized and summarized using a robust multiarray average. From the normalized data, genes with significant evidence for differential expression were identified by fitting a weighted analysis of variance (ANOVA) model using the Bioconductor limma package. We first fit linear model (specifically an ANOVA) to the gene expression data and by incorporating weights we could smoothly up- or down-weight different samples based on similarities between the sample and others of the same type. P values were calculated with a modified t test in conjunction with an empirical Bayes method to moderate the SE of the estimated log-fold changes. Genes were considered significantly differentially expressed at an unadjusted P value < .05 and a 2-fold difference.
Validation of cDNA microarray by quantitative reverse transcriptase polymerase chain reaction
Quantitation of messenger RNA (mRNA) levels was performed by the Center on Human Development and Disability Genomics Core Laboratory (University of Washington, Seattle, WA) using fluorogenic 5’ nuclease-based assays and has been previously described.
Self-contained gene set test
A self-contained gene set test was performed on 3 gene sets that were hypothesized to be affected by uterine distension (2 terms from the Gene Ontology Biological Process, “angiogenesis” and “muscle development,” as well as the Reactome myogenesis pathway) using the mroast function from the Bioconductor limma package. A self-contained gene set is based on the null hypothesis that no genes are differentially expressed against the alternative hypothesis that ≥1 genes are differentially expressed. To assess significance, the null distribution is estimated via random rotations of the orthogonalized residuals from the ANOVA model.
Ingenuity Pathway Analysis
We used the Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems, Redwood City, CA) to discover pathways and transcriptional networks in the gene expression microarray data. Our data set containing gene identifiers and corresponding expression changes between the experimental groups and P values was uploaded into the IPA application. Each identifier was mapped to its corresponding object in the Ingenuity knowledge base. The functional analysis identified the biological functions and/or diseases that were most significant to the data set. Genes from the data set with >1.5-fold differential expression (up-/down-regulation) and P < .05 that were associated with biological functions and/or diseases in the Ingenuity knowledge base were considered for the analysis. The categories “top molecular and cellular functions,” “physiological systems,” “top canonical pathways,” and “top transcription factors” were primarily used in this analysis. Right-tailed Fisher exact test was used to calculate a P value determining the probability that each biological function and/or disease assigned to that data set is due to chance alone. IPA also allows prediction of the activation or inhibition of transcription factors involved in the gene expression patterns seen in our study.
For experiments involving amnion epithelial cell cultures, the methods have previously been described for procurement of intact human fetal membranes from term elective cesarean deliveries before labor and cell culture. Preterm human amnion and myometrial tissues from women with (N = 4) and without (N = 36) polyhydramnios delivered by cesarean delivery were obtained. Polyhydramnios was defined as excessive amniotic fluid as measured by an amniotic fluid index >24 cm. In the group with polyhydramnios, the women were in early preterm labor (maximum cervical dilation of 3 cm), but the fourth was not in labor. Control tissues were obtained from women without polyhydramnios, who delivered preterm before the onset of labor (N = 36) for other indications (preeclampsia, intrauterine growth restriction). The mean gestational age of the women with polyhydramnios and preterm controls was similar (32.3 ± 1.4 vs 33.1 ± 2.7 weeks).
The human amnion and myometrial samples from women with twins were obtained at the time of cesarean delivery from 15 women not in labor and 10 women in early preterm labor. The myometrial tissues were collected from the upper aspect of the lower uterine segment immediately after hysterotomy. Preterm labor was defined as the presence of regular uterine contractions with evidence of cervical dilation, which ranged from 1-3 cm. The mean gestational age of the women with twins in preterm labor and not in labor were similar (34.3 ± 1.6 vs 35.4 ± 1.2 weeks).
RNA extraction, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assays have been previously published. Primer sequences for angiotensin II receptor type 1 ( AGTR1 ), ephrin type-B receptor 1 ( EPHB1 ), heparin-binding epidermal growth factor-like growth factor ( HBEGF ), membrane metalloendopeptidase ( MME ), and transforming growth factor, beta-induced ( TGFBI ) are shown in Supplemental Table . Cytokine and chemokine levels in the myometrial and amnion tissues were determined by Luminex multiplex technology using commercially available human cytokine/chemokine kits (Biorad, Hercules CA). The number of cases and controls used in the polyhydramnios or twins pregnancy protein and mRNA analyses varied slightly from the total cohort due to availability of tissues for both analyses. Final numbers for each group are noted in the figure legends.
In vitro stretch of human amniocytes
Static stretch was applied to the amnion cell monolayers using a Flexercell strain unit (FX-4000; Flexcell International Corp, McKeesport, PA). The stretch unit consists of a computer-controlled vacuum unit and a BioFlex base plate (to hold the 6-well culture dishes; Flexcell International Corp., Burlington, NC) and gaskets, BioFlex loading stations, and BioFlex plates. The base plates are placed in a humidified incubator with 5% carbon dioxide at 37°C. The system applies strain using a vacuum to deform the flexible membrane on which cells are cultured.
The BioFlex culture plate used was a 6-well culture plate with an optically clear, thin membrane (0.02-in thick) of rubber coated with collagen type I. The BioFlex loading stations are designed to provide uniform radial and circumferential strain across the membrane surface up to 20% elongation. They are composed of a single plate and 6 individual, removable planar cylinders (6 loading posts) per plate. The membrane is pulled over the post by the vacuum; each radius is stretched by the same amount at any point in the circle thus providing uniform “equibiaxial” strain for all cells on the membrane.
Confluent human amnion epithelial cells were plated on flexible type I collagen coated plates and were subjected to static stretch and 11% elongation. An 11% stretch was the maximum amount of stretch that could be used without the amnion cells being lifted off the culture plates and dying. Nonstretched cells were used as controls and cultured under identical conditions but were not subjected to mechanical stimulation. Time periods of up to 6 hours of stretch were used for the studies. After 6 hours of stretch, >90% of cells were viable as measured by trypan blue exclusion and >99% of cells remained attached to the 6-well culture plates after stretch protocols.
RNA extraction, qRT-PCR assays, and primer sequences for IL-1β, IL-8, and glyceraldehyde 3-phosphate dehydrogenase have been previously published. Primer sequences for TNF and IL-6 are shown in Supplemental Table . Note that IL-8 mRNA levels due to stretch have been previously reported by our group, but our work here represents independent experiments not previously published.
Study outcomes were quantities of uterine activity, cytokines/chemokines, and prostaglandins in amniotic fluid (IL-1β, TNF-α, IL-6, IL-8, CCL2, PGE 2 , PGF 2α ). For uterine activity, we specifically compared mean 24-hour hourly contraction area in the first 10 days after balloon inflation to isolate the effect of balloon-induced uterine strain from confounding effects associated with advancing gestation. We used analysis of covariance to assess differences between postinoculation peak values for control and balloon groups with adjustment for each animal’s baseline value immediately prior to balloon inflation. P -values were based on analysis of covariance models adjusting for baseline fit on log-transformed values. A Wilcoxon rank sum test was used for the mRNA qRT-PCR related to the microarray analyses. All of these statistical analyses were conducted using Intercooled STATA 8.2 for Windows 2000 (StatCorp, College Station, TX). Ratios for mRNA or protein levels to glyceraldehyde 3-phosphate dehydrogenase between unstretched and stretched cells were assessed by Wilcoxon signed rank test (nonparametric test for related samples) using SPSS (Version 10.0; IBM Corp, Armonk, NY). Significance was considered at P < .05. Adjustment for multiple comparisons was not performed given the limited sample size.
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council and the Weatherall Report, “The Use of Nonhuman Primates in Research.” The protocol was approved by the University of Washington Institutional Animal Care and Use Committee (permit no. 4165-01). All surgery was performed under general anesthesia and all efforts were made to minimize suffering. Human fetal membranes and myometrial samples were obtained from women with approval from the local ethics committee (Regional Research Ethics Committee 6283, London-Chelsea Ethics Committee 10/H0801/45, approved Dec. 23, 2010). Informed consent was obtained from patients before the collection of tissues.
Uterine activity, cytokines, and prostaglandins
As this was the first study of uterine overdistention in a nonhuman primate, balloon inflation protocols varied as we gathered more information about the effects of uterine distention on preterm labor. Initially, a slow inflation protocol was tried, but when labor did not occur after 18 days, a more rapid inflation protocol was used for the remaining experiments. Experimental results correlating balloon volume, labor, and days until delivery are shown in Table 1 . The mean gestational age at the time of saline inoculation or balloon inflation was similar (saline 135 days, balloon 133 days) corresponding to approximately 80% of a typical Macaca nemestrina gestation in our colony (term ∼172 days). The uterus was quiescent in all 11 animals prior to balloon inflation or saline inoculation. Within 10 days of balloon inflation, preterm labor occurred in 3 of 4 animals experiencing the greatest uterine distention due to inflation of 3 balloons. There was a notable outlier of a single animal delivering at term despite inflation of 3 intraamniotic balloons (animal no. 3) ( Table 1 ). Intrapartum fetal demise occurred in 2 of 3 animals after inflation of 3 balloons. In 1 case, this was due to an obvious cord entanglement with the intraamniotic catheters (animal no. 4) ( Table 1 ). In the second case, the cause was unknown, but presumably was due to cord compression by the inflated balloon (animal no. 5) ( Table 1 ). Our results suggest that greater uterine distention is associated with preterm labor, but progression to term in some cases may occur with a similar degree of uterine stress.
Temporal relationships among uterine activity, amniotic fluid cytokines, and prostaglandins are depicted in 1 animal after saline inoculation and 2 following balloon inflation ( Figure 1 ). Saline infusion in either the choriodecidual space or the amniotic fluid of controls was not associated with an elevation in amniotic fluid cytokines ( Figure 1 A). Rapid balloon inflation was associated with significant elevations of IL-1β, TNF-α, IL-8, CCL2, PGE 2 , and PGF 2α in the amniotic fluid following balloon inflation and preceding labor ( Figure 1 B, and Table 2 ) (IL-1β, P = .003; TNF-α, P = .02; IL-6, P = .02; IL-8, P = .001; CCL2, P = .007; PGE 2 , P = .001; PGF 2α , P < .001). The temporal profile of amniotic fluid cytokine and prostaglandin elevations more closely resembled an “inflammatory pulse” than a sustained and building inflammatory event typical of infection-associated PTB in nonhuman primates. Levels of PGE 2 and PGF 2α in the amniotic fluid remained slightly higher following the pulse than at baseline.
|Saline infusion (N = 5)||Balloon inflation (N = 6)||Saline infusion||Balloon inflation, no labor (N = 2) a||Balloon inflation, labor (N = 3)||Saline vs balloon, labor|
|IL-1β||0.003 (0.001)||0.03 (0.02)||0.005 (0.002)||1.3 (1.1)||3.7 (3.3)||.003|
|TNF-α||0.01 (0.009)||0.02 (0.008)||0.02 (0.01)||0.4 (0.3)||0.9 (0.4)||.02|
|IL-6||4.3 (1.4)||8.4 (3.7)||10.7 (2.2)||28.4 (21.2)||78.3 (25.9)||.02|
|IL-8||0.8 (0.2)||8.9 (3.8)||1.5 (0.3)||13.6 (10.2)||26.9 (2.4)||.001|
|CCL2||1.4 (1.0)||1.9 (0.9)||3.0 (2.0)||5.3 (3.8)||9.4 (3.5)||.007|
|PGE 2||0.7 (0.6)||0.09 (0.02)||0.7 (0.5)||0.4 (0.3)||1.3 (0.4)||.001|
|PGF 2α||0.3 (0.06)||0.3 (0.09)||0.7 (0.1)||0.7 (0.2)||2.9 (1.2)||<.001|
The peak levels of cytokines and prostaglandins correlated with the volume of uterine distention. Small volumes of balloon inflation induced a small rise or no change in cytokines and prostaglandins and were not associated with labor. There were no significant differences in amniotic fluid cytokines or prostaglandins between the balloon group not in labor by 10 days and controls. In 1 animal undergoing slow distention, a rise in IL-8 was only evident after greater volumes were added to the balloon in the final 2 days before cesarean delivery ( Figure 1 C). These results illustrate that preterm labor after balloon inflation is associated with higher levels of amniotic fluid proinflammatory cytokines, chemokines, and prostaglandins. The pattern of a rise in amniotic fluid cytokines, chemokines, and prostaglandins following a uterine stress induced by balloon inflation and preceding labor suggests that inflammation may be a trigger of parturition for this type of uterine stress. The observation that smaller volumes of balloon inflation can also induce a rise in cytokines also suggests that there is an inflammatory threshold, which must be reached to trigger labor.
Fetal plasma cytokines were available at the time of cesarean delivery in 2 cases after balloon inflation (animals no. 2 and 6) and in 3 saline controls ( Table 3 ). In both cases of uterine overdistention, fetal IL-1β, TNF-α, IL-6, and IL-8 were markedly higher than in saline controls. In the 2 animals with uterine overdistention, fetal IL-6 levels met criteria for the fetal inflammatory response syndrome (>11 pg/mL). In contrast, peak levels of maternal plasma cytokines (IL-1β, TNF-α, IL-6, IL-8) were similar between controls and balloon cases.
|Experiment no.||Fetal plasma cytokines a|
a Values are pg/mL. Peak values for saline controls and values at delivery for uterine overdistention animals. Fetal cytokines were only available from 3 saline controls and 2 animals undergoing uterine overdistention. Balloon animals no. 2 and 6 correspond to same experiments numbered in Tables 1 , 2 , and 4 .
Biomechanical modeling of uterine wall stress in the nonhuman primate model
To investigate the biomechanical effects of the varying balloon inflation protocols on uterine wall stress, we used the law of Laplace to approximate the relationship between intrauterine pressure and wall stress. We also took into account the small variations in size of the fetus and volume of amniotic fluid in each experiment to improve our estimates. In our calculations, we assumed the starting intrauterine pressures among the animals were the same. The calculated wall stress before balloon inflation differed at most by 5.4%, suggesting that large changes in wall stress after balloon inflation are not highly dependent on the initial resting pressure. After a brief equilibration period following balloon inflation, we calculated that pressures increased between 3.4 torr (animal no. 1) and 6.8 torr (animal no. 4) ( Table 4 ). This translates to increases of uterine wall stress between 40-83%. From these calculations, it appears that some animals experienced a modest increase in uterine stress (animals no. 1, 2, and 6), and 3 animals experienced a much larger increase (no. 3, 4, and 5) ( Table 4 ). The largest increase in uterine wall stress was associated with development of preterm labor in 2 of 3 cases.
|Experiment no.||Before balloon inflation||After balloon inflation||Labor within 10 d|
|Intrauterine pressure||Wall stress||Deviation from average wall stress||Intrauterine pressure||Pressure increase||Wall stress||Increase wall stress|
|Pa (torr) a||N/m 2 × 10 3||%||Pa (torr) a||%||N/m 2 × 10 3||%|
|1||1600 (12)||134||5.4||2049 (15.4)||28||187||40||No|
|2||1600 (12)||128||1.1||2112 (15.8)||32||186||45||No|
|3||1600 (12)||127||0||2398 (18.0)||50||220||73||No|
|4||1600 (12)||123||–3.2||2500 (18.8)||56||224||83||Yes|
|5||1600 (12)||123||–2.8||2420 (18.2)||51||215||75||Yes|
|6||1600 (12)||126||–0.5||2145 (16.1)||34||187||48||Yes|
a Pa is an International System of Units measure of pressure and stress defined as 1 N/m 2 ; torr is a unit of pressure that is approximately equal to 1 mm Hg and is presented as this may be more intuitive for clinician.
Uterine and placental histopathology
Histopathology of uterine biopsies (lower uterine segment and fundus) and the placenta was reviewed to determine if balloon distention was associated with injury of either the myometrium or fetal membranes. Normal uterine tissue and placental histology was noted in all controls and balloon animals (data not shown). Rare individual perivascular neutrophils and lymphocytes were seen in uterine sections of both groups, without evidence of alteration of architecture or stromal changes. Placental histology was consistent with changes normally seen in late gestation without evidence of abruption or tissue necrosis. This suggests that labor in the balloon group was not the result of gross injury to the fetal membranes or myometrium.
Cytokine response in the amnion after in vitro stretch and in women with polyhydramnios
To determine if uterine stress was also associated with cytokine expression and secretion in vitro, we examined the longitudinal expression of labor-associated genes in human amniocytes up to 3 hours following stretch. IL1β, IL6, and IL8 mRNA levels were significantly increased vs nonstretched controls at multiple time points following amniocyte stretch ( P < .05) ( Figure 2 ). There was no increase in TNF mRNA expression at any of the time points up to 6 hours after stretch. To further validate our observations, we investigated amnion tissues from women with polyhydramnios, in which acute (or subacute) uterine overdistention is thought to occur. Amnion tissues were obtained from women with polyhydramnios (N = 3) and preterm controls not in labor (N = 36) delivered by cesarean. There was a significant increase in mRNA expression of TNF ( P < .001) and IL6 ( P < .05), but not IL1β or IL8 in the amnion from women with polyhydramnios ( Figure 3 ). In summary, inflammatory cytokine mRNA expression is increased after stretch of human amniocytes in vitro and in the amnion of women with polyhydramnios, which supports the data observed in the nonhuman primate model.
Cytokine and chemokine protein levels in the amnion and myometrium of women with twins
To determine if inflammatory cytokine and chemokine proteins were increased in women with twins, we tested matched samples of amnion and myometrium from women with twins in early labor compared to twins not in labor. We chose twin pregnancies in early labor, because we hypothesized that these cases had crossed a threshold of distention necessary to trigger labor and would manifest a greater myometrial inflammatory response than twins not in labor at the same gestational age. TNF-α was significantly increased in the amnion of women with twins in early labor ( P < .01) ( Figure 4 ). In myometrial samples from women in early labor, IL-6, IL-8, and CCL2 levels were significantly elevated (all P < .05) ( Figure 4 ). Both amnion and myometrial tissues contribute to inflammation in women with twins in early labor, but the cytokine profile from each tissue appears to be distinct.