Objective
We assessed the efficacy of a maternal multidose azithromycin (AZI) regimen, with and without antiinflammatory agents to delay preterm birth and to mitigate fetal lung injury associated with Ureaplasma parvum intraamniotic infection.
Study Design
Long-term catheterized rhesus monkeys (n = 16) received intraamniotic inoculation of U parvum (10 7 colony-forming U/mL, serovar 1). After contraction onset, rhesus monkeys received no treatment (n = 6); AZI (12.5 mg/kg, every 12 h, intravenous for 10 days; n = 5); or AZI plus dexamethasone and indomethacin (n = 5). Outcomes included amniotic fluid proinflammatory mediators, U parvum cultures and polymerase chain reaction, AZI pharmacokinetics, and the extent of fetal lung inflammation.
Results
Maternal AZI therapy eradicated U parvum intraamniotic infection from the amniotic fluid within 4 days. Placenta and fetal tissues were 90% culture negative at delivery. AZI therapy significantly delayed preterm delivery and prevented advanced fetal lung injury, although residual acute chorioamnionitis persisted.
Conclusion
Specific maternal antibiotic therapy can eradicate U parvum from the amniotic fluid and key fetal organs, with subsequent prolongation of pregnancy, which provides a therapeutic window of opportunity to effectively reduce the severity of fetal lung injury.
Maternal genital tract infections are an important and potentially preventable cause of preterm birth, especially the birth of the most immature infants. Among bacterial pathogens, genital mycoplasmas play a unique role in causing or contributing to adverse obstetrical outcomes at virtually every stage of pregnancy. Isolation of Ureaplasma spp from the placental membranes and amniotic fluid has been consistently associated with histologic chorioamnionitis, preterm birth, and adverse perinatal outcomes. Ureaplasma spp are the most common microorganisms isolated from the amniotic fluid, cord blood, respiratory tract, and cerebrospinal fluid (CSF) of infants born prematurely who develop bronchopulmonary dysplasia (BPD)/chronic lung disease and neurodevelopmental disabilities. There is compelling evidence from human studies and experimental models that in utero infection with Ureaplasma spp elicits a sustained and dysregulated immune response, which is a harbinger of the BPD phenotype.
For Editors’ Commentary, see Contents
We have previously demonstrated in a rhesus monkey model that U parvum as the sole pathogen in the amniotic cavity elicits a robust inflammatory response associated with chorioamnionitis, a progressive increase in uterine activity culminating in preterm labor and delivery and fetal lung injury. Similarly, there are clinical reports indicating that Ureaplasma infections during pregnancy elicit intense inflammatory responses in the fetus, amniotic cavity, and mother. The extent of the inflammatory response and severity of fetal lung damage (progressive alveolitis and bronchiolitis) were related to the duration of in utero infection. Intraamniotic Ureaplasma infection alters fetal lung development and augments a proinflammatory, profibrotic response in the preterm lung exposed postnatally to ventilation and hyperoxia. Despite in vitro susceptibility of Ureaplasma spp to erythromycin, trials of erythromycin therapy in the first 4 weeks of neonatal life in Ureaplasma -colonized preterm infants fail to demonstrate efficacy in preventing BPD or eliminating respiratory tract colonization. To prevent chronic lung disease/BPD it may be necessary to eradicate Ureaplasma spp with antenatal antibiotic therapy to forestall the additive effects of fetal lung injury with postnatal ventilation and/or high oxygen exposure. Although there are reports of maternally administered erythromycin for the eradication of intraamniotic infection (IAI), there is little agreement in the literature regarding the effectiveness of erythromycin to prevent preterm delivery or eliminate intraamniotic Ureaplasma infections during pregnancy.
Despite the large body of evidence that links maternal subclinical infection with premature labor, there is controversy as to whether antenatal antibiotic intervention inhibits preterm labor, prolongs gestation, or otherwise improves perinatal outcomes. A prevailing attitude of therapeutic nihilism regarding the role of antibiotics in the prevention of prematurity or neonatal sequelae may be unwarranted given that conflicting results of these clinical trials may reflect shortcomings and/or variations in study design. Among confounders are the inclusion of women with preterm contractions without infection, administration of antibiotics at varying stages of intrauterine infections, and antibiotics that do not target the appropriate pathogen. Alternatively, antibiotics alone without the addition of antiinflammatory agents may not effectively treat chorioamnionitis or reduce the proinflammatory mediators that play a key role in the initiation of labor. There is now a critical need to study specific antibiotic and antiinflammatory regimens for defined pathogens under experimental conditions to evaluate placental transfer and pharmacokinetics (PK) and to establish biological plausibility for antenatal treatment of Ureaplasma infections in utero.
The primary objectives of the current study were to assess the efficacy of antenatal azithromycin (AZI) in the treatment of experimental IAI with U parvum and in mitigating the extent of fetal lung injury in utero. AZI is a 15-member azalide, a subclass of macrolide antibiotics with structural similarity to erythromycin, but with a prolonged duration of action, improved tissue penetration, and extended range of coverage against Ureaplasma spp. We compared U parvum IAI alone (untreated animals) with the effects of maternal AZI treatment alone or in combination with the antiinflammatory drugs dexamethasone (DEX) and indomethacin (INDO) on eradicating U parvum IAI, modulating proinflammatory mediators, inhibiting preterm uterine activity, delaying premature birth, and ameliorating fetal lung injury.
Materials and Methods
Animal model
Study protocols were approved by the Institutional Animal Care and Utilization Committee and guidelines for humane care were followed. Timed-pregnant rhesus monkeys ( Macaca mulatta, n = 23) were adapted to a vest and mobile catheter protection device. Intrauterine surgery was then performed at 119 days of gestation (range, 115–127 days) to implant fetal electrocardiogram electrodes, and catheters in the amniotic fluid, maternal femoral vein, and artery. Intravascular catheters were implanted in the fetal jugular vein, and carotid artery in a select number of fetuses (n = 2) for the analysis of AZI concentrations in the fetal circulation following maternal treatment. Postoperative infusions of cefazolin sodium to prevent infection and tocolytic medications to control uterine irritability (eg, terbutaline sulfate and/or atosiban) were prescribed as previously published. Tocolytic medications were discontinued when uterine quiescence was achieved and at least 48 hours before experimental procedures.
Microorganism
U parvum (serovar 1), a low passaged clinical isolate, was originally recovered in pure culture from the placenta of a woman with chorioamnionitis who delivered a preterm infant with Ureaplasma sepsis. The isolate was initially serotyped by immunoblotting with monoclonal antibodies and subsequently the Ureaplasma spp and serovar was determined by real-time polymerase chain reaction (PCR). The organisms were grown in 10B broth (Thermo Scientific, Rockford, IL), frozen, and stored in aliquots at –80°C until utilized for experimental protocols. Inoculum sizes were determined by assessing the number of colony-forming units (CFU)/mL in thawed samples and making the appropriate dilutions to deliver the desired number of organisms.
Experimental design
Animals were allocated to the study by random assignment from the Oregon National Primate Research Center colony, and treatment and control (IAI untreated) were performed concurrently. Intraamniotic inoculation was performed on approximately day 128 of gestation (range, day 124–135; n = 16) with U parvum 7-14 × 10 7 CFU/mL. A subset of animals then received either maternal AZI treatment alone (12.5 mg/kg, every 12 h, intravenous [IV] for 10 days; n = 5) or in combination with DEX (4 mg/kg/d, IV for 4 days) and INDO (100 mg/d, orally [PO] for 5 days; n = 5). Antenatal antibiotic/antiinflammatory treatments commenced after 6-8 days of U parvum IAI and with a concomitant increase in uterine activity and/or cervical effacement/dilation over a 24-hour observational period, as determined by a modified Bishop score. The modified Bishop score assessed the following 4 components upon vaginal examination: cervical consistency, cervical length, cervical dilation, and fetal station. Each component was scored 0-3, with a maximum score of 12. A modest increase in uterine activity (3000-5000 hourly contraction area [HCA]) and/or a Bishop score of >5 was utilized as an indicator for the commencement of treatment. Primary comparisons were made between the AZI treatment groups and IAI untreated animals, which served as treatment controls. Additional historical controls were included for comparative evaluation of placental and fetal lung histopathology between U parvum IAI untreated and antimicrobial treatment groups. These control animals received either a single intraamniotic bolus infusion of sterile sucrose phosphate buffer (n = 3) or physiologic saline (n = 4) in place of U parvum on day 135 of gestation (range, 131–138 days). Poststudy antibiotic treatment (Zithromax; Pfizer, New York, NY: 500 mg, PO for 1 day; 250 mg, PO for 4 days) was administered to ensure clearance of the organisms from each animal prior to its return to the colony.
Sampling protocol for AZI PK
Following the initial dose of maternal AZI, an intensive sampling protocol was followed in which paired samples were taken from maternal plasma and amniotic fluid at 0 (to indicate a trough sample before infusion), 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 hours, then every 12 hours thereafter during 10 days of maternal AZI administration. The intensive sampling protocol was repeated after the final dose of AZI and sampling was continued periodically throughout the washout phase to compare maternal plasma and amniotic fluid AZI concentrations with results from the initial sampling period. To assess the placental transfer of AZI to the fetal circulation, samples of fetal plasma were obtained from 2 animals during the initial maternal AZI dosing and subsequently all fetal cord blood samples were paired with maternal plasma samples at delivery. Transfer between maternal and fetal compartments was estimated using the percentage of maternal plasma concentrations. PK analyses were performed by a 2-compartment model (utilizing forced function, ADAPT II pharmacokinetic/pharmacodynamic systems analysis software) to determine the rate of transplacental transfer into the amniotic fluid compartment and half-life as previously described.
Uterine activity, preterm labor, and cesarean section
Intraamniotic pressure was continuously recorded from the time of surgery, digitized, and analyzed as previously described, with the addition of a new data acquisition system (PowerLab ML880 16/30P; LabChartPro software v6.1.2; ADInstruments, Colorado Springs, CO). The integrated area under the intrauterine pressure curve was used as the measure of uterine activity and reported as the mean of the cumulative hourly contraction area (HCA, mmHg.sec/h) over 24 hours. Preterm labor was defined as HCA >8000 mmHg.sec/h for >2 consecutive hour epochs and with associated changes in cervical effacement, dilation (modified Bishop score >8, determined by vaginal examination over a 24-hour observational period), or preterm premature rupture of membranes. Cesarean section was performed to optimize the collection of intact gestational tissues when vaginal delivery was considered imminent (as defined above).
Amniotic fluid cytokines, prostaglandin E 2 and F 2α , and leukocytes
Beginning 48 hours before IAI, amniotic fluid was sampled daily until delivery. Samples were centrifuged and the supernatant frozen and stored at –20°C as previously described. Quantities of interleukin (IL)-1β, -6, and -8; tumor necrosis factor-alpha; prostaglandin (PG) E 2 ; and PGF 2 α were determined using commercially available human- or rhesus monkey–specific enzyme-linked immunosorbent assay kits (BioSource International, Camarillo, CA) and enzyme immunoassay kits (Cayman Chemical, Ann Arbor, MI) previously validated for use in the rhesus monkey. Amniotic fluid leukocytes from centrifuged cell pellets were counted daily using a hemocytometer after erythrocyte lysis in 2% glacial acetic acid.
AZI concentrations
AZI (ng/mL) was quantified from 100 μL of plasma or amniotic fluid using a liquid-liquid extraction method coupled with high-performance liquid chromatography tandem mass spectrometry detection. A standard curve was prepared in either plasma or amniotic fluid with AZI concentrations (range, 6–3000 ng/mL). Quality control samples were prepared by spiking plasma with AZI for final concentrations of 18, 180, and 1800 ng/mL and erythromycin (internal standard, 100 ng/mL). AZI was extracted from all unknown samples, standards, and quality control samples by addition of 2 mL of methyl-tert-butyl ether. Separation of mobile phase analytes was achieved using reverse-phase high-performance liquid chromatography with an isocratic run on an XTerra C8 column (Waters Corp, Milford, MA). Acceptance criteria for the assay mandated that the back-calculated values for curve and quality control standards were within ±15% of nominal concentration. All intercoefficients and intracoefficients of variation were <10%.
Culture and PCR analysis
Quantitative cultures and PCR for U parvum were performed on serial samples of amniotic fluid and maternal whole blood obtained before and after inoculation, and on fetal cord blood at cesarean section delivery. PCR and cultures were also performed on fetal CSF, pieces of the lower right lobe of the lung, fetal brain, fetal membranes, and placenta. Fetal membrane culture swabs were collected as described previously. To ensure each animal was free of natural Mycoplasma infections before inoculation, and to guarantee clearance of U parvum from each animal prior to its return to the colony, maternal nasal and vaginal swabs for PCR and culture for genital mycoplasmas were taken at prestudy and poststudy time points. Periodic amniotic fluid cultures were obtained for facultative and anaerobic bacteria using standard bacteriological procedures.
Samples of fluids, blood, and tissues were frozen immediately upon collection at –80°C until transported on dry ice to the Diagnostic Mycoplasma Laboratory at the University of Alabama at Birmingham. Once received, specimens were processed quantitatively using 10B broth media and A8 agar as described previously. U parvum colonies were identified on A8 agar by urease production in the presence of calcium chloride indicator. Real-time PCR assays were performed on batched specimens containing all samples from a single animal using primers as described previously by Xiao et al.
Histopathology
After cesarean section, fetuses were euthanized by barbiturate overdose followed by exsanguination and necropsy performed for tissue collection. Fetuses and placentas were examined, weighed, and evaluated for abnormalities against gestational age-matched controls. Fetal and maternal surfaces of placenta and chorioamnion were assessed for integrity, clarity, coloration, and infarction. Fetal tissues were collected as previously described, fixed in neutral buffered formaldehyde, embedded in paraffin, stained with hematoxylin-eosin, and subsequently sectioned at 5 μm for standard histologic examination by a trained pathologist (T.K.M.).
Histologic evaluation of the fetal lung and placental membranes
Fetal lung hematoxylin-eosin tissue slides were reviewed by 3 investigators blinded to the experimental conditions. Microscopic lung lesions were semiquantitatively scored from 0-3, for the presence and extent of alveolar macrophages, neutrophils, and lymphocytic infiltration of alveolar walls, using a modified method from Viscardi et al. The scoring was determined as: 0 = none; 1 = the presence of polymorphonuclear cells and mild lymphocytic infiltration of alveolar walls; 2 = focal increases in both mononuclear and polymorphonuclear cells in alveoli; and 3 = extensive inflammation in both alveoli and terminal bronchioles, exuberant proliferation and sloughing of type II pneumocytes, diffuse interstitial collections of mononuclear cells with multiple peribronchiolar lymphocytic aggregates, and hyperplasia of the overlying epithelium.
Multiple representative areas of the gross placenta and fetal membrane were sampled for histologic examination and scored for signs of IAI, defined as the presence of neutrophils marginating into the membranes as described by Romero et al. A negative score indicated no neutrophils present.
Statistical analysis
Data are presented as mean ± SEM. Data were first examined for normality by the Kolmogorov-Smirnov test and the Levene median test for equal variance, and where necessary, data were transformed by natural logarithm to equalize variance. Outcomes of selected gestational ages (ie, at surgery, inoculation, and delivery) and experimental intervals (ie, from inoculation to treatment, to delivery, and to peak amniotic fluid U parvum colony counts) were analyzed by 1-way analysis of variance (ANOVA) followed by Tukey all pairwise comparison test. The intervals from time to 0 (CFU/mL) following the onset of AZI treatment were compared by t test. For statistical analysis of uterine activity, the mean cumulative 24-hour HCA (mm Hg.sec/h) was used and compared by 1-way ANOVA between all experimental groups (IAI alone, AZI alone, and AZI plus DEX/INDO). Similarly, amniotic fluid proinflammatory mediators (PGE 2 , PGF 2 α , cytokines) and leukocytes were statistically compared using 1-way ANOVA on repeated measures, followed by Tukey test for all pairwise comparisons on combined baseline values before inoculation (n = 16) vs combined peak concentrations during IAI (n = 16). Minimum values during AZI alone or AZI plus DEX/INDO 10-day treatment protocol were compared to concentrations just prior to the start of treatment by Student paired t test. Statistical analysis was conducted using software (SigmaStat, version 2.0; Jandel Scientific Software, San Francisco, CA), and significance was accepted at P < .05.
Results
Inoculation-to-delivery interval and gestational length
There is evidence for a clear and uniform prolongation of in utero fetal survival after antimicrobial treatment, with a mean inoculation-to-delivery interval of 20.9 ± 1.4 days vs 13.7 ± 2.5 days, respectively ( P < .05), and a 2- to 3-fold increase in the percentage of undelivered animals at 18-20 days after inoculation compared to untreated animals with IAI ( Figure 1 ). A significant increase in the inoculation-to-delivery interval was noted in animals treated with AZI alone compared to untreated IAI animals (22.4 ± 2.0 days vs 13.7 ± 2.5 days, respectively), while there were no significant differences between AZI plus DEX/INDO and IAI animals (19.4 ± 1.5 days vs 13.7 ± 2.5 days, respectively). No difference was observed between the AZI- and AZI plus DEX/INDO–treated animals (22.4 ± 2.0 days vs 19.4 ± 1.5 days, respectively), therefore data were combined for statistical purposes and illustration in Figure 1 . The average gestational age at intraamniotic inoculation with U parvum was 128 days (range, 124-135 days) and did not differ statistically among groups: U parvum alone, 130 ± 2.4 days (n = 6); U parvum with AZI, 127 ± 1 day (n = 5); and U parvum with AZI plus DEX/INDO, 128 ± 2 days (n = 5). The mean inoculation-to-treatment interval was 6.2 ± 0.8 days (range, 6–8 days) and did not differ significantly between AZI alone and AZI plus DEX/INDO treatment groups (6.0 ± 0.8 days vs 6.4 ± 0.9 days, respectively). While the primary criterion for the initiation of treatment was 6-8 days of infection to provide consistency, only animals that also had a concomitant increase in uterine activity and/or change in cervical dilation (as determined by a modified Bishop score over a 24-hour observational period) were included in the analysis. All animals receiving antibiotics (with or without antiinflammatory agents) completed the 10-day regimen, except for 1 monkey, which completed 5 days of AZI plus DEX/INDO treatment, followed by premature rupture of membranes and cesarean section delivery at 137 days of gestation. All treated and untreated animals delivered live fetuses. Despite prolongation of in utero survival by antimicrobial therapy and an average delay in preterm birth of >7 days (range, 5–15 days), treated animals delivered approximately 2 weeks before term (149 ± 1.8 days) compared to instrumented historical saline or media control animals (161 ± 1.5 days or 154 ± 2.5 days, respectively).
Uterine activity, amniotic fluid cytokines, PGs, and leukocytes
Before intraamniotic inoculation the uterus was quiescent with the average 24-hour HCA <300 mmHg.sec/h. Following U parvum IAI there was a progressive increase in uterine contractions that reached mean peak levels of 1295 ± 228 mmHg.sec/h within 6-8 days and/or prior to the initiation of antibiotic treatment ( Table 1 ) ( P < .05; n = 16). U parvum IAI also led to a significant influx of leukocytes into the amniotic fluid, and up-regulation of proinflammatory cytokine concentrations (tumor necrosis factor-alfa, IL-1β, -6, and -8) and PGE 2 and PGF 2 α ( Table 1 ) ( P < .05; n = 16). Both AZI alone and AZI plus DEX/INDO treatment significantly reduced amniotic fluid concentrations of proinflammatory mediators compared to pretreatment levels ( Table 1 ) ( P < .05). A greater reduction was observed with AZI plus DEX/INDO treatment than with AZI alone, especially in regard to PG and cytokine concentrations ( Table 1 ). However, the significant inhibitory effect on proinflammatory mediators (in both groups) was confined to the 10-day treatment interval. Subsequently, within several days (range, 1–12 days) after cessation of treatment, we observed a secondary rise in amniotic fluid leukocytes and proinflammatory mediators (data not shown), coinciding with an increase in uterine activity that culminated in preterm labor and cesarean section delivery.
| Variable | Ureaplasma parvum (n = 16) | AZI alone (n = 5) | AZI plus DEX/INDO (n = 5) | |||
|---|---|---|---|---|---|---|
| Baseline | Peak concentration | Pretreatment concentration | Posttreatment concentration | Pretreatment concentration | Posttreatment concentration | |
| UA (mmHg.sec/h) | 268 ± 31 | 1295 ± 228 a | 969 ± 423 | 347 ± 120 b | 1451 ± 438 | 285 ± 47 b |
| PGE 2 (pg/mL) | 83 ± 13 | 5830 ± 1800 a | 2821 ± 1458 | 1145 ± 535 b | 3810 ± 1541 | 191 ± 135 b |
| PGF 2 α (pg/mL) | 84 ± 12 | 2099 ± 480 a | 1372 ± 368 | 556 ± 212 b | 2358 ± 1213 | 166 ± 42 b |
| TNF-alpha (pg/mL) | 31 ± 20 | 1255 ± 374 a | 1227 ± 441 | 39 ± 26 b | 927 ± 155 | 22 ± 13 b |
| IL-1β (pg/mL) | 30 ± 16 | 859 ± 243 a | 209 ± 98 | 25 ± 16 b | 786 ± 251 | 6 ± 5 b |
| IL-6 (ng/mL) | 5 ± 2 | 98 ± 27 a | 146 ± 54 | 19 ± 10 b | 167 ± 34 | 9 ± 2 b |
| IL-8 (ng/mL) | 1 ± 1 | 56 ± 20 a | 81 ± 52 | 30 ± 24 b | 97 ± 22 | 14 ± 5 b |
| Leukocytes (cells/μL) | 25 ± 6 | 3015 ± 601 a | 1840 ± 675 | 594 ± 248 b | 2796 ± 571 | 540 ± 129 b |
a Significant compared to baseline preinfection ( P < .05, n = 16);
b significant compared to pretreatment ( P < .05, n = 5 each AZI group).
AZI concentrations
The temporal relationships of maternal plasma and amniotic fluid AZI concentrations (ng/mL) with U parvum colony counts (CFU/mL) in a representative animal are illustrated in Figure 2 . Data collected after the initial dose of AZI demonstrate maternal plasma mean peak levels of 3239 ± 520 ng/mL were achieved within 1 hour, while corresponding mean peak amniotic fluid AZI concentrations reached 56.2 ± 25 ng/mL within 7 hours. Frequent sampling from 2 fetuses demonstrated peak plasma levels of 85.5 ± 9.5 ng/mL within 1.2 hours of the initial maternal AZI dose, indicating a placental transfer rate of approximately 2-3%. Multiple AZI dosing (12.5 mg/kg, every 12 h, IV for 10 days) resulted in sustained trough levels of 75-300 ng/mL in the maternal circulation and amniotic compartment. At steady-state, inhibitory concentrations of AZI in the amniotic fluid equaled or exceeded maternal plasma levels at the completion of the 10-day treatment protocol and during the washout phase, suggesting accumulation and an extended therapeutic window within the amniotic compartment ( Figure 2 ). A slow decay in the amniotic fluid AZI washout occurs and is likely due to extended tissue penetration. The estimated half-life after multiple dosing of AZI was 2.6 days in maternal plasma and 7.5 days in the amniotic fluid. At cesarean section delivery, fetal umbilical cord blood AZI levels were approximately 18% of maternal plasma levels (51.5 ± 20.6 ng/mL vs 291 ± 158.4 ng/mL, respectively; n = 6).
U parvum cultures and PCR analysis
All animals were screened by culture and PCR for naturally occurring genital mycoplasmas prior to inoculation and results were all negative. Periodic amniotic fluid cultures for facultative or anaerobic bacterial infections remained negative throughout the study. None of the mothers with U parvum IAI had positive blood cultures ( Table 2 ), were febrile (with rectal temperature >38.9°C), or had systemic leukocytosis. After inoculation of U parvum (7-14 × 10 7 CFU/mL), and initial dilution with amniotic fluid, there was an exponential growth of microorganisms that peaked at 1.91 × 10 6 CFU/mL within 3 days (range, 1–8 days) and stabilized thereafter at 1.09 × 10 6 CFU/mL ( Figure 3 ) prior to antibiotic treatment. Amniotic fluid U parvum (CFU/mL) peaked in each animal prior to the commencement of antibiotic treatment and was rapidly reduced by maternal AZI administration within 24 hours ( Figure 3 ). Intraamniotic U parvum infection was cleared within 4 days (range, 2–7 days; n = 10) after repeated maternal AZI dosing, irrespective of adjunctive antiinflammatory treatments ( Figure 3 ). There was no significant difference in the inoculation-to-peak (CFU/mL) intervals between animals or the time to 0 (CFU/mL) from the start of antibiotic treatment.
| Fluids and tissues | Ureaplasma parvum (n = 6) | Combined AZI treatment groups (n = 10) | ||
|---|---|---|---|---|
| Culture (%) | PCR (%) | Culture (%) | PCR (%) | |
| Amniotic fluid | 6/6 (100) | 6/6 (100) | 1/10 c (10) | 7/10 (70) |
| Maternal blood | 0/6 (0) | 1/6 (17) | 0/10 (0) | 0/10 (0) |
| Fetal blood | 0/6 (0) | 1/6 (17) | 0/10 (0) | 1/10 (10) |
| Fetal membranes | 6/6 (100) | 6/6 (100) | 1/10 (10) | 4/10 (40) |
| Placenta | 6/6 (100) | 6/6 (100) | 1/10 (10) | 2/10 (20) |
| Fetal lung | 5/6 (83) | 6/6 (100) | 1/10 (10) | 5/10 (50) |
| Fetal brain | 0/5 (0) | 0/3 (0) b | 0/10 (0) | 1/10 (10) |
| Cerebrospinal fluid | 0/4 (0) a | 1/4 (25) b | 0/9 (0) | 1/9 (11) |
a Fetal cerebrospinal fluid cultures were not uniformly obtained due to contamination with blood;
b PCR results were inconclusive in 2 of 5 brain samples;
c One AZI plus dexamethasone/indomethacin–treated animal experienced regrowth of U parvum at delivery despite clearance of amniotic fluid by culture and PCR during 10-treatment interval.
In the absence of antimicrobial therapy, fetal lung, placenta, fetal membranes, and amniotic fluid were 83-100% positive for U parvum by culture and PCR at the time of delivery ( Table 2 ). Fetal cord blood was positive by PCR but not culture in 1 of 6 fetuses (17%). Fetal brain and CSF remained negative by culture but CSF tested positive in 1 of 4 fetuses (25%) by PCR. The PCR results in 2 of 5 fetal brain samples from U parvum –infected animals were inconclusive due to unresolved assay inhibitors, and in 2 fetuses CSF samples were not obtained or were contaminated by blood. At delivery, amniotic fluid samples and placental and fetal tissues from treated animals were negative by culture in 90% of cases ( Table 2 ) (n = 10). However, 1 animal treated with AZI plus DEX/INDO presented with regrowth of U parvum at delivery despite clearance of microorganisms during the antibiotic treatment protocol (as determined by negative culture and PCR), suggesting the possibility of persistent organisms in biofilm. Some treated animals were PCR positive but culture negative and may reflect the fact that DNA from nonviable organisms may still amplify for variable periods of time.
Histopathology of the fetal lung and placental membranes
Fetuses infected in utero with U parvum demonstrate a progressive alveolitis and bronchiolitis ( Figure 4 ). An exudative pneumonia develops within days after infection characterized by a diffuse presence of neutrophils and macrophages in the alveolar spaces, a stimulation of type II pneumocytes and areas of necrosis in alveolar and terminal airway epithelium. In more prolonged Ureaplasma infection (>10 days) the acute inflammatory response is followed by lymphocytic infiltration of alveolar septae, prominent hyperplasia and hypertrophy of type II pneumocytes, reactive proliferation of epithelial cells, and an apparent thickening of alveolar walls ( Figure 4 , B). In longer-term infection (>15 days) there is also the conspicuous development of peribronchiolar lymphoid tissue aggregates accompanied by hyperplasia and hypertrophy of bronchiolar epithelium ( Figure 4 , C). Maternal AZI treatment initiated after 6-8 days of U parvum IAI (irrespective of antiinflammatory treatments) substantially reduced the magnitude and distribution of intraalveolar leukocytes, albeit scattered foci of amniotic debris and clumps of leukocytes remained ( Figure 4 , D). There was also a posttreatment reduction in alveolar wall leukocytic infiltration, in type II pneumocyte hyperplasia, and in the overall appearance of alveolar wall thickness. It was notable that maternal AZI treatment prevented the development of peribronchiolar lymphocytic aggregates which were absent or rare in AZI-treated animals, and if present were much smaller and not accompanied by mucosal hyperplasia of the bronchiolar epithelium.
