1. Steven Lovrich, Gundersen Lutheran Medical Foundation ASSOCIATION BETWEEN MYCOPLASMA INFECTION AND COMPLICATIONS DURING PREGNANCY

2.  Mollicutes: “soft skin”  Intracellular parasite  Lack cell wall  Trilayered external membranes  2 genera: Mycoplasma  14 human species; three pathogenic  M. hominis  M. genitalium  M. pneumoniae Ureaplasma  2 human species; both pathogenic  U. urealyticum  U. parvum (Taylor-Robinson et al., An International Journal of Obstetrics and Gynecology, 2010) MYCOPLASMA Microscopic view of Mycoplamsas

3.  Facultative anaerobes  Pleomorphic  Limited genome  Unable to gram stain  Culture? (Larsen et al., Infectious Diseases in Obstetrics and Gynecology, 2010) LABORATORY CHARACTERISTICS

4.  Normal Flora/Non-pathogenic colonizers?  Pathogenic?  Opportunistic pathogen  Location of colonization  Host immune response  Conditions of pregnancy  Co-infections  Genetic factors  Environmental factors VIRULENCE

5.  Adherence to host cell by mycoplasmal adhesion proteins/lipoproteins  Stimulate secretion of pro-inflammatory cytokines (tumor necrosis factor- , interleukin, & interferon-γ)  Stimulate release of prostaglandins which leads to protease production  Protease can cause adverse pregnancy outcomes  miscarriage, pre-term labor, bacterial vaginosis, chorioamnionitis, spontaneous abortion, perinatal morbidity & mortality, PROM, etc. PATHOGENICITY DURING PREGNANCY

6. Problems:  Genital tract infections associated with approximately 50% of preterm deliveries  13% of pregnancies in the U.S. result in preterm delivery or low infant birth weight  60% of mortality among infants (with no anatomic/chromosomal defects) is low birth weight (Kataoka, Journal of Clinical Microbiology, 2006), (Taylor-Robinson, An International Journal of Obstetrics and Gynecology, 2010) PRE-TERM BIRTH

7.  Strongly associated with:  Chorioamnionitis  Pelvic inflammatory disease  Bacterial vaginosis  Pregnancy  Lower gestational age at delivery  Lower birth weight  Increased neonatal morbidity & mortality  Increase risk for miscarriage at 14 weeks  Infant  Pneumonia MYCOPLASMA HOMINIS Mycoplasma hominis on agar plate

8.  Causative agent of urethritis  Associated with cervicitis, PID, and endometritis in women  Pregnancy- Unknown (Taylor-Robinson, An International Journal of Obstetrics and Gynecology, 2010) MYCOPLASMA GENITALIUM Electron micrograph of M. genitalium

9.  In 2002, U. urealyticum & U. parvum distinguished as separate species  Therefore, studies pre-2002 confounded UREAPLASMA SPP.

10.  Colonization of placenta= increases risk for fetal & maternal inflammation  Increase risk of preterm labor  Increase risk for miscarriage (@14 weeks)  Vertically transmitted to fetus potentially causing:  Bacteremia  Pneumonia  Chronic lung disease  Nervous system infections UREAPLASMA UREALYTICUM Electron micrograph of U. urealyticum

11.  Vertically transmitted to fetus in utero or during delivery  Bacteremia, pneumonia, chronic lung disease, & nervous system infections  More prevalent in amniotic fluid of preterm pregnancies than U. urealyticum  If colonization occurs can cause:  PROM  Preterm labor  Chorioamnionitis (in mother)  Early onset sepsis & BPD (bronchopulmonary dysplasia) in baby (Larsen et al., Infectious Diseases in Obstetrics and Gynecology, 2010) UREAPLASMA PARVUM Electron micrograph of U. parvum

12. Objectives: 1. Determine if colonization of either Ureaplasma species had association with miscarriage or preterm labor 2. To perfect detection methods and discrimination of species  Methods: Tested 239 pregnant women (PCR) from the La Crosse area for colonization with Ureaplasma urealyticum & parvum during early prenatal period ASSOCIATION OF UREAPLASMA WITH PRETERM BIRTH

13.  239 patient samples at start  192 follow ups at Gundersen Lutheran  47 lost  27 adverse events  23 preterm birth (≤36 weeks) or miscarriage  4 preterm labor (stopped)  Significance of Colonization  P-value ≤ 0.05 SUMMARY OF RESULTS

14. RESULTS Bacterial characteristics and cause of early delivery for the preterm birth group. Presence (+/-) of: Cause of Subject Gestational wk at delivery U. parvum U. urealyticum preterm delivery a 1 6 + - Miscarriage 2 9 + - Miscarriage 3 18 + - Miscarriage 4 26 + - PROM 5 30 + - Preeclampsia 6 31 - - PROM 7 34 + - FSUA 8 35 + + Preeclampsia 9 35 + - Preeclampsia 10 35 + - FSUA 11 35 + - FSUA 12 35 + - FSUA 13 36 + - FSUA 14 36 + - FSUA 15 36 - + FSUA 16 36 - - FSUA 17 36 - - FSUA 18 36 + - FSUA 19 36 + - FGR 20 36 + - Preeclampsia 21 36 + - Preeclampsia 22 36 - - FSUA 23 36 + - FSUA a PROM, premature rupture of the membranes; FSUA, failure to suppress uterine activity; FGR, fetal growth restriction.

15. RESULTS 27 abnormal pregnancy outcomes -25 associated with U. parvum U. parvum strongly associated with their occurrence (p=0.003)

16.  Previous study  Small population sample  Little diversity & limited risk factors Parameters:  Project collaboration with WiNHR (Wisconsin Network for Healthcare Research)  4 different hospital sites: 200 samples per site Aurora Health, Gundersen Lutheran, UW-Hospital (Madison), Marshfield Cinic  Test for 4 Mycoplasma species * Objective: To determine if any of the 4 Mycoplasma species correlate with pregnancy abnormalities or adverse outcomes  Examine multiple risk factors CURRENT STUDY

17. Normal healthy pregnant women targeted in 4 sites across Wisconsin Swabs of urogenital tract (~12 weeks) Samples blinded DNA extracted and forwarded to Gundersen Lutheran Polymerase Chain Reaction (PCR) Hybridization assay EXPERIMENTAL DESIGN

18. HYBRIDIZATION ASSAY Aminated probe (4 species specific probes used) Biotinylated amplification product

19. HYBRIDIZATION ASSAY Aminated probe (4 species specific probes used) Biotinylated amplification product

20. HYBRIDIZATION ASSAY E Aminated probe (4 species specific probes used) Biotinylated amplification product Strepavidin-HPO conjugate

21. HYBRIDIZATION ASSAY E(S) Aminated probe (4 species specific probes used) Biotinylated amplification product Strepavidin-HPO conjugate Substrate

22.  PCR hybridization assay completed on samples  Correlations between species and adverse pregnancy outcomes STUDY PROGRESS

23.  Thanks to:  Microbiology Research Laboratory  Gundersen Lutheran Medical Foundation  Dr. Steve Callister  Dean Jobe ACKNOWLEDGEMENTS