ASSOCIATION BETWEEN MYCOPLASMA INFECTION AND COMPLICATIONS DURING PREGNANCY

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

MYCOPLASMA 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 Microscopic view of Mycoplamsas § 2 human species; both pathogenic § U. urealyticum § U. parvum (Taylor-Robinson et al. , 2010) An International Journal of Obstetrics and Gynecology,

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

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

PATHOGENICITY DURING PREGNANCY 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.

PRE-TERM BIRTH 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)

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

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

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

UREAPLASMA UREALYTICUM 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 Electron micrograph of U. urealyticum

UREAPLASMA PARVUM 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: Electron micrograph of U. parvum § PROM § Preterm labor § Chorioamnionitis (in mother) § Early onset sepsis & BPD (bronchopulmonary dysplasia) in baby (Larsen et al. , Infectious Diseases in Obstetrics and Gynecology, 2010)

ASSOCIATION OF UREAPLASMA WITH PRETERM BIRTH 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

SUMMARY OF RESULTS 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

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

RESULTS • 27 abnormal pregnancy outcomes -25 associated with U. parvum • U. parvum strongly associated with their occurrence (p=0. 003)

CURRENT STUDY Previous study § Small population sample § Little diversity & limited risk factors Parameters: Project collaboration with Wi. NHR ( Wis consin Ne tw ork for He al thc are 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

EXPERIMENTAL DESIGN 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

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

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

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

STUDY PROGRESS § PCR hybridization assay completed on samples § Correlations between species and adverse pregnancy outcomes

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