Understanding the Etiology of Premature Rupture of

MembranesAmy P. Murtha, MD

Vice Chair for Research

Obstetrics and Gynecology

Preterm Birth

Preterm birth defined as delivery prior to 37 weeks gestation

11.9% of all deliveries in the US 500,000 pregnancies in the US per year More than newly diagnosed breast cancer cases

Preterm Birth in the US

14

12

10

8

6

4

2

01990 1992 1994 1996 1998 2000 2002 2004 2006 2008

Year

Late preterm (34 - 36 weeks)

Early preterm (< 34 weeks)

Preterm (< 37 weeks)

2011

Preterm Birth in the US Relative to the World – WHO Data*

Country Preterm Birth Rates

Africa 11.9

North America 10.6

Asia 9.1

Central America 9.1

South America 7.9

Australia/New Zealand 6.4

Europe 6.2*Beck et al 2010: http://www.who.int/bulletin/volumes/88/1/08-062554/en

Preterm Birth in North Carolina

National Center for Health Statistics, Final Natality Data

Preterm PROM

Rupture of membranes prior to 37 weeks without labor

2-3% of all pregnancies are result of PPROM

20% of all perinatal deaths

PPROM Management

34-37 weeks Delivery

<34 weeks Admitted for duration of pregnancy

Monitored for evidence of infection or labor

Clinical detection of infection limited

Maternal fever increases risk of neonatal morbidity

Research Objectives

Impact the management of PPROM

Understand the etiology of PPROM

Biomarkers of Intrauterine Infection

Prospective cohort studydaily blood samplesmonitored for infectionreceived antibiotics and steroids

Subjects divided into those with and without funisitis (infection in umbilical cord)

PPROM Study

FunisitisN=54

No Funisitis

N=53P value

% Clinical Chorioamnionitis

22/54 (41%) 1/49 (2%) <.0001

GA at delivery (wks) mean (SD)

28.5 (3.2) 31.5 (2.6) <.0001

Birthweight (grams) 1182 (516) 1716 (547) <.0001

PPROM to delivery interval (mean days)

12.7 (10.3) 16.4 (14.7) .11

Median Serum IL-6 (pg/mL)

Time point FunisitisNo

Funisitis P value

24-48 hours 7.5 (1.8–48.5)

2.8 (0.0–9.6)

<.0001

48-72 hours 5.3 (0.1-55.4)

1.5 (0.0-19.9)

<.0001

>72 hours 2.9 (0.1-42.0)

1.8 (0.0-22.4)

.03

24-72 hours 5.3 (0.1-55.4)

1.9 (0.0-19.9)

<.0001

Infection and Inflammation in PregnancyBlood and Tissue Repository

Over 400 subjects enrolled with over 4,000 samples collected

140 complete sets with daily blood samples collected

Detailed delivery samples collected, barcoded and stored

Maternal and cord blood, placenta, fetal membranes, umbilical cord, buccal swab

Understanding the Etiology of PPROM

Mechanisms resulting in membrane thinning (weakening)

Focus on the chorion cell layer

Amnion

Chorion

Decidua

Etiology of PPROM

Poorly understood and difficult to study

Infection and inflammation important in development of PPROM

Fetal Chorion Layer

Accelerated apoptosis in chorion layerPreterm and term in presence of histologic chorioamnionitis1

Thinned or absent chorion among PPROM subjects2

1 Murtha AP et al. Infect Dis Obstet Gynecol 2002;10:93–962 George RB et al. Am J Perinatol 2008;25:29–32

PPROM and the Chorion

Amnion

Chorion

Amnion

Chorion absent

Normal PPROM

Moving forward…prospective collection

• Fetal membrane chorion thickness atmembrane rupture

distant from rupture

Bacterial presence in fetal membranes membrane rupture

distant from rupture

Primary cell culture and cell line

Methods – Data Collection

46 paired membrane samples collectedRupture site (rupture) or overlying cervix

Distant to rupture site (distant)

IHC Methods – Sample Processing

Chorion and choriodecidual thickness measured using Image J® software

Chorion

Choriodecidua

FISH Methods – Sample Processing

Bacteria identified using fluorescence in situ hybridization (FISH) with Alexa Red probe to 16s rRNA subunit

Custom oligonucleotide sequence 5´-F-ACTGCTGCCTCCCGTAGGAGTTTATTCCTT (Invitrogen®)

DistantRupture

Chorion

Term, No Labor

10X magnification 10X magnification

Distant

Chorion

Rupture

PPROM

10X magnification10X magnification

PPROM Preterm Labor

Preterm, No Labor

Term Labor

Term, No Labor

0

100

200

300

400

RuptureDistant

Chorion Thickness by Clinical Group

Mea

n T

hick

ness

(µ

m)

*

^

* P = 0.0024 ^ P = 0.0015

Number of Bacteria per Membrane Quadrant by Clinical Group

PPROM Preterm Labor

Preterm, No Labor

Term Labor

Term, No Labor

0

20

40

RuptureDistant

*P < 0.0005 ^P = 0.002

Num

ber

Bac

teria

Per

Qua

dran

t

^

*

PPROM Preterm Term0

10

20

30

40

RuptureDistant

Number Bacteria per Membrane Quadrant Without Chorioamnionitis

Num

ber

Bac

teria

Per

Qua

dran

t *P = 0.03 ^P = 0.006

N = 7 N = 14 N = 16

^

*

What else….

Bacterial DNA sequencing from fetal membranes Ureaplasma parvum as a pathogen in pregnancy Thrombin’s role in fetal membrane integrity Cigarette smoke exposure and effect on membranes and

placenta Progesterone’s role in modulating the inflammatory

response MMP activity and regulation of progesterone Mechanisms of progesterone function through non-

nuclear PR (PGRMC1)

Clinical Research in OB/GYN MFMU Clinical Trials Network (Federal) Antenatal Late Preterm Steroids (ALPS): A

Randomized Placebo-Controlled Trial DMID vaccine trials (Federal) National Children’s study (Federal) Newborn Epigenetic Study (NEST) (Federal) Children’s Environmental Health Initiative (Federal) 17OH PC for Prevention of Recurrent Preterm Birth

(Industry) Labor induction with misoprostil (Industry) Wound Vac in Obesity (Industry) Prenatal diagnosis with Maternal free fetal DNA

(Federal/Industry) The effects of SCDs on fibrinolysis in patients

undergoing Cesarean Delivery (Industry) A Prospective Study of the Hemostatic Differences

between Women with and without Vaginal Bleeding in Pregnancy (Federal)

An Intervention to Maximize Research Sample Collection for Pregnant Subjects at Delivery (Duke)

Comparing Focused Ultrasound and Uterine Artery Embolization for Uterine Fibroids (Federal)

Bridging Interdisciplinary Research Careers in Women’s Health (BIRCWH) K23 (Federal)

Pelvic Floor Clinical Trials Network (Federal) Management of Menorrhagia in Women with Bleeding

Disorders (Federal) Neural Prosthetic Control of Continence and

Micturition (Federal) Effectiveness Of Sacrospinous Ligament Fixation

(SSLF) versus Uterosacral Ligament Suspension (Federal)

Cervical Intraepithelial Neoplasia Cohort Study (Federal)

Ambulatory Treatments for Leakage Associated with Stress (Federal)

Anticholinergic vs. Botox Comparison study: The ABC Trial (Federal)

Outcomes Following Vaginal Prolapse Repair and Mid Urethral Sling (OPUS) Trial (Federal)

Prospective randomized study of sperm production in healthy volunteers receiving pregabalin or placebo. (Federal/Industry)

GIS Health Data in the Blanchard Neighborhood of Haiti: Assessment of Community Needs Guide Community Public Health Practice? (Foundation)

Molecular Markers of Human Sperm Function (Federal)

Questions?