Steven Lovrich, Gundersen Lutheran Medical Foundation

ASSOCIATION BETWEEN MYCOPLASMA INFECTION

AND COMPLICATIONS DURING PREGNANCY

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

Facultative anaerobes

Pleomorphic

Limited genome

Unable to gram stain

Culture?

(Larsen et al., Infectious Diseases in Obstetrics and Gynecology, 2010)

LABORATORY CHARACTERISTICS

Normal Flora/Non-pathogenic colonizers?

Pathogenic?

Opportunistic pathogen Location of colonization Host immune response Conditions of pregnancy Co-infections Genetic factors Environmental factors

VIRULENCE

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

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

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

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

In 2002, U. urealyticum & U. parvum distinguished as separate species

Therefore, studies pre-2002 confounded

UREAPLASMA SPP.

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

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

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

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

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 deliverya

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.

Table 2. Association between urogenital infection with ureaplasmas and miscarriage, preterm

labor controlled by treatment, and/or preterm birth.

Pregnancy outcome (no.) No. (%) U. urealyticum p value No. (%) U. parvum p value Normal (165) 16 (10) NA 84 (51) NA Abnormal (27) 2 (7) 0.999 22 (81) 0.003

Miscarriage (3) 0 0.999 3 (100) 0.245 Preterm birth (20) 2 (10) 0.999 15 (75) 0.057 Preterm labor (4) 0 0.999 4 (100) 0.122

RESULTS

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

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

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

HYBRIDIZATION ASSAY

Aminated probe(4 species specific probes used)

Biotinylatedamplification product

HYBRIDIZATION ASSAY

Aminated probe(4 species specific probes used)

Biotinylatedamplification product

HYBRIDIZATION ASSAY

E

Aminated probe(4 species specific probes used)

Biotinylatedamplification product

Strepavidin-HPO conjugate

HYBRIDIZATION ASSAY

E(S)

Aminated probe(4 species specific probes used)

Biotinylatedamplification product

Strepavidin-HPO conjugate

Substrate

PCR hybridization assay completed on samples

Correlations between species and adverse pregnancy outcomes

STUDY PROGRESS

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

ACKNOWLEDGEMENTS