Abstract

1. HOW DO UREAPLASMA MICROORGANISMS INVADE THE PROTECTED ENVIRONMENT OF THE BABY? Kim Newman, MAT1, Victoria HJ Roberts, PhD2, Peta L Grigsby, PhD2 1Portland Christian High School, Portland, Oregon; 2Division of Reproductive Science, Oregon National Primate Research Center, Beaverton, OR Abstract Objective: To investigate the transmigration of U.parvum across intact gestational membranes and measure the immunological response of the amnion and choriodecidual membranes to the microorganisms. • Finding optimal growth media for U.parvum while maintaining tissue healthwas challenging. Our data supports that tissue is most healthy (for 48 hours) in DMEM with 10% fetal calf serum and 1% L-glutamine (-Ab/Am) (Figure 6). However, U.parvum did not grow in any of the media conditions tested (Table 2). Introduction: Almost 13% of all pregnancies deliver preterm and as a result the preterm infant often has life-long health complications which may include lung dysfunction and neurologic impairment.1 Several factors contribute to preterm birth, with about 30-40% of these cases attributable to intrauterine infections.2 The predominate bacteria is U.parvum.The aim of this study is to use an in vitro model to investigate the transmigration of U.parvum across intact gestational membranes and measure the immunological response of the amnion and choriodecidual membranes to the microorganisms. Methods: Gestational membranes were mounted on a Transwell ® culturesystem, which allowed us to test the choriodecidua side and the amnion side of the membranes individually. In order to test the chorioamniotic membranes using the Transwell ® system, several difficulties needed to be addressed; which media(s) was conducive to both U.parvum growth and tissue health. U.parvum growth was tested using quantitative culture and PCR analysis at the Diagnostic Mycoplasma Laboratory (Alabama). The viability of the tissue was assessed through a colorimetric assay using tetrazolium salts added to the culture medium. The long-term objective of this project will be to stimulate the membranes with U.parvum (107 cfu/mL) and examine the inflammatory response via cytokine and prostaglandin production from the component tissue layers. Results: We tested 4 media conditions: 2-SP (sucrose phosphate); DMEM (-Ab/Am) [Dulbecco Modified Eagle Medium supplemented with 10% fetal calf serum and 1% L-glutamine, without antibiotics and antimycotics], DMEM (-Ab/Am), 2-SP with AF (amniotic fluid), and DMEM (+Ab/Am). The viability of the membranes evaluated by a colorimetric assay using tetrazolium salts initially show that the membranes are healthiest in 2-SP:DMEM (-Ab/Am). The procedures need to be repeated to validate these results. U.parvum cultured in each of the media conditions tested produced no viable bacterialcolonies. Conclusions: Further conditions need to be evaluated to optimize the growth of U.parvum in the Transwell ® culture system. Once we have identified the optimal media for both U.parvum and the gestational tissue, we aim to investigate how the bacteria crosses this protective barrier and what impact it has on facilitating the immunological response driving preterm labor. Table 2:U.parvum colonies were not found in any of the growth media after being subcultured. The 2SP:DMEM- may have been a “broth only” positive or less than 10 organisms/mL. PCR analysis indicated that the U.parvum was present but dead. • The invasion properties of U.parvum will be measured through intact chorion and amnion membrane tissue layers in a Transwell ® in vitro culture system (Figure 3).6 • Tissue Acquisition and Transwell ® Culture Preparation • Chorioamniotic membranes were obtained within 10 minutes of elective cesarean delivery from healthy term pregnancies after obtaining written consent (OHSU). • The membranes were cut 2-3 cm from the placental disc, rinsed in sterile Hank’s Balanced salt solution, and transported to the laboratory in Dulbecco Modified Eagle Medium (DMEM). • Segments representing all layers of the membranes were cut into 2 X 2 cm squares and attached with a sterilized silicone rubber ring to the Transwell® culture system (Figures 3 and 4). Figure 3:The gestational membranes were held together with silicone rubber rings in the upper chamber of a Transwell ® device. The choriodecidual region occupies the upper chamber, while the amnion region, the lower chamber . 6 Figure 6 • Alternative Strategies and Future Directions • Our future trouble-shooting may include allowing the U.parvum to grow into the log phase before culturing in the various media. • Along with the previously tested media, we may include 10B broth, a known culture media for U.parvum. The pH will need to be adjusted to maintain tissue viability. Introduction • Almost 13% of all pregnancies in the US deliver preterm (Figure 1). Infants born preterm (< 37 weeks) can have a variety of life-long health complications which may include: lung dysfunction and neurologic impairment, including cerebral palsy. 1 • Several factors contribute to preterm birth, with about 30-40% of these cases attributable to intrauterine infections.2 The presence of infection triggers a cascade of events that can lead to preterm labor (Figure 2). • During ascending infection the choriodecidua is the first-line barrier in contact with pathogens that can cross the membranes and infect the amnion and amniotic fluid. Localized inflammatory responses in vivo are likely to weaken the structural integrity of the chorioamnion and allow a less virulent microorganism to also breech this barrier, such as U.parvum.8 • Indeed, clinical studies have shown U.parvum can be cultured in the amniotic fluid as early as 16-20 weeks of gestation.8Despite this, there is no direct experimental evidence demonstrating that U.parvum can breech the intact chorioamniotic membranes and gain entry into the amniotic cavity. • With the in vitro Transwell ® culture system using intact chorioamniotic tissue layers, we hope to discover how U.parvum crosses the membranes and what impact it has on the immunological response. • Is there a time threshold or a microbial concentration threshold? • Does U.parvum “piggy-back” on other bacteria? • Are the membranes weakened by other factors that allow U.parvum access to the amniotic fluid and ultimately to the baby? • What is the inflammatory response with infection? National Preterm Birth Rate Figure 4:After the membranes are attached the excess tissue is removed. Figure 5: 12-Well Transwell ® set up Discussion and Questions Results Methods • Babies develop in a protected environment surrounded by multiple gestational membranes. This creates a water-tight barrier surrounding the fetus and amniotic fluid. These membranes are the first line of defense in protecting the fetus from infection, and are comprised of three fused layers (See figure 2 insert): • 1. The amnion, an epithelial fetal layer that has direct contact with the amniotic fluid; • 2. The chorion, the outer fetal membrane layer; and • 3. The decidua which is of maternal origin and is adjacent to the uterine wall. Figure 1 • 1 mL of growth media was added to each well of a 12-well plate, and 0.5 mL of growth media was added to the upper chamber of the Transwell ® insert (Figure 5). • The membranes were incubated in 5% CO2 @ 37 °C for 24 hours. During that time, the medium was changed twice: After the first 2-4 hours and 1-2 hours prior to inoculation with U.parvum (107cfu/mL). Clinical Impact: Through understanding the pathophysiology and pathogenesis of preterm birth as a result of ascending uterine infection, strategies can be developed for early diagnosis and treatment. This in turn would decrease the incidence of preterm labor and increase the health of newborn children. • As a woman goes into labor, these membranes secrete prostaglandins, cytokines and hormones that trigger cervical dilation, membrane rupture and uterine contractions. 3 • Studies suggest that some bacteria can breech this protected environment through an ascending route from the cervical-vaginal canal. 4 • 50% of these microorganisms found in the infected amniotic fluid are Ureaplasma species (i.e., U. parvum). These organisms are typically found in normal vaginal flora of ~80% of healthy pregnant women.5 • However, there is no direct experimental evidence demonstrating that U.parvum can breech the intact chorioamniotic membranes and gain entry into the amniotic cavity, which raises several questions: • What causes the overgrowth of U.parvum in a subset of women? • How is it getting in the amniotic cavity? • Trouble Shooting • The Transwell® set-up has not been used with U.parvum;a difficult bacteria to culture. As a result, our initial investigations were devoted to evaluating the optimal bacterial growth conditions, while also maintaining the viability of the tissue. • (a) Bacterial Growth Conditions: • In duplicate, U.parvum and 1 mL of experimental media was added to a 12-well culture plate as summarized in Table 1. IV III • Thank you to: • The Murdock Foundation for funding this mind-stretching and inspirational opportunity. • Drs. Peta Grigsby and Victoria Roberts for allowing me to be their “side kick,” for their great patience with teaching me, and for allowing me to be part of the scientific process. • Dr. Leo Pereira for creating the opportunity to obtain gestational tissue and for allowing me to observe the miracle of life. • Amanda Alexander for coming along side of me teaching me valuable skills. • Melinda Murphy for going above and beyond helping us set-up our CO2 incubator. • Joel Ito for proofing and printing this poster for me. • Supported by NIH Grant HD055053 and RR000163 Acknowledgements II I • The U.parvum was incubated in 5% CO2 @ 37 °C for 24 hours. • For each media condition, 100 µL aliquots in 10B culture tubes and PCR transport buffer were frozen at -80 °C prior to shipment to the Diagnostic Mycoplasma Laboratory. • (b)Tissue Viability Studies: • Tissue viability was evaluated by a colorimetric assay using tetrazolium salts (MTT) added to the different culture media conditions. This was processed in a separate set-up where the salts were observed changing from yellow to blue assessing the tissue’s overall metabolism.7 • Tissue U. parvum Inoculation • During the summer of 2011, the following methods will be used: • 107 cfu/mL U.parvum will be added to the Transwell ® inserts on the choriodecidual side . • After 24 hours of incubation, the media will be collected from both chambers, centrifuged @ 5,000 rpm for 3 minutes at 4 °C to precipitate U.parvum. • The bacteria-free media will then be tested for cytokines and prostaglandins, while the U.parvum pellet will be sent to the Diagnostic Mycoplasma Laboratory for quantitative culture and PCR analysis. Table 1 • Figure 2: Anatomy of gestational membranes and ascending intra-uterine infection and the progressive stages of intra-uterine infection: • Stage I: Changes in vaginal or cervical flora; Stage II: Bacteria ascend from cervical-vaginal canal into the choriodecidual space, leading to an inflammatory response and choriodeciduitis; Stage III: Bacteria migrate into the amniotic cavity; Stage IV: Bacteria gain access to the fetus which can result in severe fetal infection. 4 References [1] Grether JK, Nelson KB: Maternal infection and cerebral palsy in infants of normal birth weight. Journal of the American Medical Association 278: 207, 1997. [2] Newton ER: Preterm labor, preterm premature rupture of membranes and chorioamnionitis. Clinical Perinatology 2005, 32:571-600. [3] Parry S, Strauss JF: Premature rupture of the fetal membranes. New England Journal of Medicine 1998, 338:663-668. [4] Romero R, Mazor M: Infection and preterm labor. Clinical Obstetrics and Gynecology 1988, 31(3):553-584. [5] Sperling RS, Schachter JS: Intra-amniotic infection in low birth weight infants. Journal of Infectious Diseases 157:113, 1988. [6] Zaga V, Estrada-Gutierrez G: Secretion of Interleukin-I beta and tumor necrosis factor alpha by whole fetal membranes depends on initial interactions of amnion or chorion with lipopolysaccharides or group B streptococci. Biological Reproduction 2004, 71:1296-1302. [7]Gerlier D, Thomasset N: Use of MTT colorimetric assay to measure cell activation. Journal of Immunological Methods 1986; 94:57-63. [8]Cassell, G.H., et al.: Isolation of Mycoplasma hominis and Ureaplasma urealyticum from amniotic fluid at 16-20 weeks of gestation: potential effect on outcome of pregnancy. Sex TransmDis, 1983. 10(4 Suppl): p. 294-302.