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EVIDENCE OF PLACENTAL HEMORRHAGE: RELATIONS WITH
PRETERM DELIVERY, POLYMORPHISMS IN VASCULAR
FUNCTION GENES, AND INTRAUTERINE INFECTION

presented by

Julia Marie Warner Gargano

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EVIDENCE OF PLACENTAL HEMORRHAGE: RELATIONS WITH PRETERM
DELIVERY, POLYMORPHISMS IN VASCULAR FUNCTION GENES,
AND INTRAUTERINE INFECTION
By

Julia Marie Warner Gargano

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Epidemiology

2009

ABSTRACT
EVIDENCE OF PLACENTAL HEMORRHAGE: RELATIONS WITH PRETERM
DELIVERY, POLYMORPHISMS IN VASCULAR FUNCTION GENES,
AND INTRAUTERINE INFECTION
By
Julia Marie Warner Gargano

Bleeding has been identified as a major etiologic pathway to preterm delivery
(PTD). We hypothesized that placental abruption may be an extreme manifestation of this
pathway, while early pregnancy vaginal bleeding and placental pathology findings may
identify early or subclinical manifestations, respectively. We aimed (1) to evaluate
multiple indicators of placental hemorrhage as potential components of a common
bleeding pathway, (2) to assess the associations between maternal gene polymorphisms in
thrombophilia and renin-angiotensin system pathways and PTD subtypes defined by
evidence of placental hemorrhage, and (3) to evaluate risk of histologic chorioamnionitis
and clinical chorioamnionitis in relation to early and late evidence of placental
hemorrhage.

A subcohort (N =1371) of pregnant women were recruited at midtrimester (15-27
weeks’ gestation) as part of a prospective cohort study (1998-2004). Data were
ascertained by interviews conducted at enrollment, detailed medical chart abstraction,
maternal blood assays and placental pathology examinations. We analyzed data on 996
black or white subcohort women who had complete placenta data and did not have
placenta previa. Data on functional polymorphisms in candidate, genes (methylene-
tetrahydrofolate reductase (MTHFR) C677T, MT HFR A1298C, Factor V Gl69lA

(Leiden, FVL), and angiotensinogen (AGT) G-6A) were available on 959 of these

women. Information derived from the gross and microscopic placental examinations
included histologic chorioamnionitis, disc-impacting blood clots, and Maternal Vascular
— Disturbance of Integrity (MV-I) scores.

Four manifestations of placental hemorrhage, i.e. placental abruption, disc-
impacting blood clots, top quintile of MV-I scores, and first trimester bleeding, differed
in their associations with some maternal characteristics and were not highly concordant
with one another. Subclinical evidence of placental hemorrhage identified through
placental pathology exams was associated with increased odds of PTD, particularly PTD
at <35 weeks, after accounting for clinically evident bleeding in a multivariable model.
Women who were heterozygous for FVL or the ACT -6 A allele were at increased risk of
PTD with evidence of placental hemorrhage, whereas they were not at increased risk of
PTD without evidence of placental hemorrhage. Placental abruption and disc-impacting
blood clots were associated with clinical chorioamnionitis, while bleeding in the first and
second trimesters was associated with the histologic chorioamnionitis, although there was
evidence of an interaction with delivery timing for histologic chorioamnionitis.

Multiple clinical and subclinical indicators of placental hemorrhage are related to
PTD. However, because associations with maternal characteristics, gene polymorphisms,
and intrauterine infection differed among measures of placental hemorrhage we conclude
that heterogeneity exists even within the “bleeding pathway.” PTD may be marked by
early or late bleeding for a number of reasons. Greater insight into bleeding-related

pathways may be achieved by incorporating information on subclinical hemorrhage.

DEDICATION

To Amelia, with love

iv

ACKNOWLEDGEMENTS

I am indebted to many people who have contributed to my doctoral education. I
have benefited from the mentorship of Dr. Claudia Holzman, who believed in my ability
to do independent research, and provided me with challenges that have pushed me do
much more than I thought possible. Dr. Pat Senagore gave her time and wisdom to assist
me in achieving a rudimentary understanding of some aspects of placental pathology. I
have benefited from the careful reading, critical comments, and challenging ideas
provided by all the members of my PhD committee: Dr. Holzman, Dr. Senagore, Dr.
Dorothy Pathak and Dr. Lynne Reuss. I am grateful for the time they have contributed to
my training.

It has been a great privilege to work on the POUCH Study. I cannot name all the
people who contributed to this endeavor, but I am aware that I stand on the shoulders of
many in presenting study results. Here, I mention a few people who have directly
contributed to this work. Dr. Karen Friderici and Katherine J erni gan were responsible for
the genetic assays employed in this study. Dr. Rachel Fisher provided valuable critical
feedback as I developed ideas that formed the foundation of this dissertation. Dr. Judith
Suess, Dr. Nazish Siddiqi, and Lynn Thelen reviewed the charts of suspected placental
abruption cases. Dr. Bertha Bullen and Crista Valentine have made the POUCH Study
run smoothly, and have provided direct assistance and moral support.

The Graduate School, the College of Human Medicine, and the Department of
Epidemiology provided financial support in the form of a dissertation completion

fellowship and funding for conference travel. In addition, many faculty members in the

Department of Epidemiology have been helpful teachers, and have provided the much-
needed commodity of encouragement. I would especially like to thank Dr. Mathew
Reeves. Mat gave me many opportunities to grow through a variety of side projects, and
also offered practical advice and help along the way.

Finally, I thank my family. My parents always encouraged me to pursue education
and helped me find ways to achieve my goals, even though my path meandered. My
parents and siblings set an example of perseverance and high standards that I hope I will
always carry with me. To my husband Joel, thank you for helping us to maintain some
normalcy in our home life in spite of the many hours I spent on this project, and thank
you for all your encouraging words. I am sure I could not have done this without you by

my side.

vi

TABLE OF CONTENTS

LIST OF TABLES .......................................................................................................... viii
LIST OF FIGURES ........................................................................................................... x
KEY TO ABBREVIATIONS ........................................................................................... xi
CHAPTER 1. BACKGROUND LITERATURE AND AIMS ............................................ 1
1.1. Preterm delivery subtypes and etiologic pathways .................................................. l
1.2. Evidence of placental hemorrhage and preterm delivery ......................................... 4
1.3. Polymorphisms in genes involved in vascular function
in relation to evidence of placental hemorrhage and preterm delivery .................... 8
1.4. Evidence of placental hemorrhage in relation to inflammation and infection ....... 20
1.5. Aims ....................................................................................................................... 32
CHAPTER 2. EVIDENCE OF PLACENTAL HEMORRHAGE AND PRETERM
DELIVERY ....................................................................................................................... 33
2. 1 . Introduction ............................................................................................................ 3 3
2.2. Methods .................................................................................................................. 34
2.3. Results .................................................................................................................... 40
2.4. Discussion .............................................................................................................. 50

CHAPTER 3. POLYMORPHISMS IN THROMBOPHILIA AND RENIN-
ANGIOTENSIN SYSTEM PATHWAYS, PRETERM DELIVERY

AND EVIDENCE OF PLACENTAL HEMORRHAGE ................................................. 55
3.1. Introduction ............................................................................................................ 55
3.2. Methods .................................................................................................................. 56
3.3. Results .................................................................................................................... 61
3.4. Discussion .............................................................................................................. 71

CHAPTER 4. CLINICAL CHORIOAMNIONITIS, HISTOLOGIC

CHORIOAMNIONITIS, AND EVIDENCE OF PLACENTAL HEMORRHAGE ........ 75
4.1 . Introduction ............................................................................................................ 75
4.2. Methods .................................................................................................................. 76
4.3. Results .................................................................................................................... 81
4.4. Discussion .............................................................................................................. 90

CHAPTER 5. SUMMARY ................................................................................................ 96

REFERENCES ................................................................................................................. 99

vii

LIST OF TABLES

Table 1.1. Placental abruption case definitions from selected epidemiologic studies ........ 6

Table 1.2. Studies reporting on associations between selected vascular function gene
polymorphisms and placental abruption, adapted from Zdoukoupoulos and Zintzaras ....11

Table 1.3. Studies comparing evidence of placental hemorrhage with clinical or
histologic evidence of infection ........................................................................................ 22

Table 2.1. Indicators of placental hemorrhage, maternal characteristics and risk factors,
overall and by preterm delivery status ............................................................................... 43

Table 2.2. Prevalence of four indicators of placental hemorrhage among women with
each of the other three indicators (weighted row percent), and odds ratios for bivariate
comparisons ....................................................................................................................... 44

Table 2.3. Prevalence (weighted row percent) of four indicators placental hemorrhage
according to selected maternal characteristics ................................................................... 45

Table 2.4. Odds ratios (OR) and 95% confidence intervals (CI) for the association
between four indicators of placental hemorrhage and preterm delivery (PTD), overall and
stratified by delivery timing (N =996) ................................................................................ 47

Table 2.5 Odds ratios (OR) and 95% confidence intervals (CI) for the association
between four indicators of placental hemorrhage and spontaneous or medically indicated
preterm delivery (PTD) ...................................................................................................... 48

Table 3.1 Race-specific minor allele frequencies for measured gene polymorphisms
among 560 white and 399 black women ............................................................................ 64

Table 3.2 Maternal characteristics of subcohort sample, and prevalence of placental
abruption, subclinical evidence of placental hemorrhage, and no evidence of placental
hemorrhage ........................................................................................................................ 65

Table 3.3 Association between vascular function genotypes (dominant models) and
preterm delivery among 560 white women and 399 black women ...................... 67

Table 3.4 Association between vascular function genotypes (dominant models) and PTD
subtypes defined by presence or absence of evidence of hemorrhage compared with term
deliveries among 560 white women and 399 black women .............................................. 68

Table 3.5 Association between vascular function genotypes (dominant models) and
preterm delivery subtypes defined by placental abruption, subclinical evidence of
placental hemorrhage, and no evidence of placental hemorrhage compared with term
deliveries among white or black women ........................................................................... 69

viii

Table 4.1. Distribution of selected maternal characteristics in study sample and
prevalence of histologic chorioamnionitis (HCA) and clinical chorioamnionitis (CCA)
according to maternal characteristics. ................................................................................ 85

Table 4.2. Association between clinical chorioamnionitis (CCA) and histologic
chorioamnionitis (HCA) overall and stratified by preterm delivery, N=996 ..................... 87

Table 4.3. Association between histologic chorioamnionitis (HCA) and unexplained high
MSAF P and early and late evidence of placental hemorrhage. ......................................... 88

Table 4.4. Association between clinical chorioamnionitis (CCA) and unexplained high
MSAFP and early and late evidence of placental hemorrhage. ......................................... 89

ix

LIST OF FIGURES

FIGURE 2.1. Venn diagram showing 15 possible combinations of the 4 indicators of
placental hemorrhage. Disc-impacting blood clot = oval, double line, microscopic
hemorrhage = rectangle, double line; first trimester bleeding = rectangle, single line;
placental abruption = oval, single line . ............................................................................. 49

PTD

OR

CI

BMI

HCA

CCA

MV-I

MSAF P

MTHF R

AGT

FVL

KEY TO ABBREVIATIONS

Preterm Delivery

Odds Ratio

Confidence Interval

Body Mass Index

Histologic Chorioamnionitis

Clinical Chorioamnionitis

Maternal Vascular — Disturbance of Integrity
Maternal Serum Alpha-fetoprotein
Methylene tetrahydrofolate reductase
Angiotensinogen

Factor V Leiden

xi

CHAPTER 1.

BACKGROUND LITERATURE AND AIMS

1.1. Preterm delivery subtypes and etiologic pathways

Preterm delivery, defined as delivery of a fetus prior to 37 completed weeks’
gestation, increases risks of neonatal morbidity and mortality(1). Approximately 10% of
singleton deliveries in the United States are preterm, and rates Of preterm delivery have
been increasing(2). Although many risk factors for preterm delivery have been identified,
including African-American race, low socioeconomic status, alterations in vaginal flora,
stress, low and high body mass index, substance abuse and short interpregnancy interval,
accurate prediction of preterm delivery is not possible(3). The strongest predictor of
preterm delivery is a history of preterm delivery in a prior pregnancy(4, 5).

Preterm delivery is an occurrence with many causes and multiple clinical
presentations(6-13). Therefore, researchers have subdivided preterm deliveries into
relatively homogeneous subtypes to improve specificity of associations with candidate
risk factors(5, 11, 14-17). Many studies, including those of the National Institutes of
Child Health and Development Maternal-Fetal Medicine Units, have classified preterm
deliveries according to gestational age at delivery(18-20). Other classification schemes
have relied on clinical presentation. For example, Meis divided preterm deliveries into
‘medically indicated’ (i.e., those associated with placenta previa, placental abruption,
antepartum hemorrhage, preeclampsia/eclampsia and renal disease regardless of whether
they were induced), and spontaneous (i.e. all other preterm deliveries), and found that
they differed with respect to their associations with maternal age, hemoglobin levels, and

bacteriuria, but were similar with respect to many other risk factors.(15) More recent

studies have restricted the ‘medically indicated’ category to those deliveries initiated by
prelabor cesarean or induction of labor at preterm gestations(5, 10). These are often
contrasted with all other preterm deliveries (i.e. ‘spontaneous’ preterm deliveries), or
spontaneous preterm labor with intact membranes, and preterm premature rupture of
membranes(10, 11, 17, 20, 21).

Subdividing preterm deliveries into subtypes can diminish statistical power
because fewer women are present in each outcome category; however, this effect may be
offset by improved specificity. Savitz et. a1. empirically assessed whether the magnitude
of associations of many well-known or “strongly suspected” risk factors differed
statistically (P<.20) between indicated and spontaneous deliveries using data from the
Pregnancy, Infection, and Nutrition (PIN) study (N=2319; 158 (55%) spontaneous, 128
(45%) indicated preterm deliveries).(10) The authors found that poverty index, clinic site,
parity, bacterial vaginosis, and body mass index differed in the magnitude of their
associations with spontaneous or medically indicated preterm births, while race, maternal
age, marital status, education, prior preterm delivery, maternal height, and smoking had
similar associations between the two preterm delivery subtypes. The authors concluded
that the choice of whether to “lump” or “split” should be made based on the study
question, balancing the loss of statistical power from excluding medically indicated births
with the gain in precision from having a more etiologically homogeneous sample.

Substantial heterogeneity exists even within these preterm delivery subtypes.
Preterm deliveries may be initiated for many maternal or fetal indications. Common
maternal indications for preterm birth include hypertensive disorders, antepartum

hemorrhage, placenta previa and diabetes(22). Common fetal indications include

intrauterine growth restriction, oligohydrarnnios, malformations, or fetal distress(22).
Causes of spontaneous preterm deliveries may be equally varied, with infection, stress,
uterine distension, nutritional factors, environmental exposures, and multiple vascular
placental problems operating along separate pathways or in tandem(6-8, 23). Some
avenues of research have focused on specific pregnancy complications (e. g. pre-
eclampsia, placental abruption, intrauterine growth restriction) and. have considered those
cases that culminate in preterm delivery as more severe or as resulting from different
causes(24, 25). Pregnancy complications may inform research into etiologic pathways
that lead to preterm delivery through subclinical manifestations of similar pathologic
processes.

In 2005, the March of Dimes published a research agenda that identified four
“major pathophysiologic pathways” to preterm delivery: infection/inflammation,
maternal/fetal stress, abruption or decidual hemorrhage, and mechanical stretch(3). The
most studied pathway to date has been infection/inflammation. Intrauterine infection
causes a significant proportion of spontaneous PTD, particularly earlier deliveries.
Bacteria ascend from the lower genital tract before or during pregnancy, infect the
membranes, and initiate an inflammatory response culminating in preterm labor or
preterm premature rupture of membranes(9). These infections, which often have no
symptoms, must be identified through sampling of amniotic fluid, fetal cord blood, or
delivered placental tissue that shows evidence of histologic chorioamnionitis (HCA)(19).
Other types of inflammation may also predispose women to preterm delivery(26).

The “abruption or decidual hemorrhage” pathway has been less comprehensively

studied. The overarching theme of this dissertation is to expand understanding of this

pathway by examining multiple sources of evidence of placental hemorrhage, including
clinical evidence and evidence derived from placental pathology examinations using data
fi'om the prospective Pregnancy Outcomes and Community Health (POUCH) Study.
Chapter 2 explores various hypothesized manifestations of placental hemorrhage and
their relations with one another, maternal characteristics, and preterm delivery. Chapter 3
focuses on functional polymorphisms in three candidate genes that have previously been
studied in relation to placental abruption, with a goal of investigating their relations with
preterm deliveries that have evidence of placental hemorrhage. Chapter 4 delves into
possible relations between evidence of placental hemorrhage and infection/inflammation
identified prior to delivery or through histopathologic examination of delivered placental
tissue. Chapter 5 provides a brief summary of the findings of chapters 2-4, and adds some

thoughts about future directions.

1.2. Evidence of placental hemorrhage and preterm delivery

Placental abruption, defined as separation of the placenta from the uterus prior to
delivery of the fetus, is a rare, potentially disastrous pregnancy complication diagnosed in
approximately 1% of all pregnancies in the United States.(27) Placental abruption is a
clinical diagnosis, typically made in response to symptoms such as pain, vaginal
bleeding, and tetanic uterine contractions(28); however, these symptoms may have other
causes, and they are not always present(29). Retroplacental hemorrhage at the separation
site can result in a hematoma, a gross pathologic lesion of abruption. Some diagnoses are
made (or confirmed) by visualization of a hematoma during an ultrasound scan(29-31); in

clinical practice, however, whether a scan is performed depends on the level of clinical

suspicion or intensity of prenatal care. Sometimes a clot adherent to the placenta is used
to diagnose or confirm abruption, but clots may be present due to normal intrapartum
bleeding. Afier delivery, placental pathologists are sometimes consulted to corroborate a
diagnosis. Pathologists typically look for evidence that a clot was present prior to
delivery, including infarcted, compressed, or otherwise affected adjacent tissue(32, 33).

Epidemiologic studies of risk factors for placental abruption have not employed
consistent case definitions (Table 1.1). Many studies have focused on clinical diagnosis
without specifying diagnostic criteria, sometimes taking diagnoses entered on birth
certificates, registries or hospital discharge databases(24, 27, 34-36). Other definitions
have required combinations of Signs and symptoms. These have included sonographic
visualization of retroplacental hematoma(37, 38), vaginal bleeding after 20 weeks or at
delivery(8, 39-42), uterine pain or tendemess(3 8, 40, 41, 43-45), and strong contractions
or increased uterine tone(3 8, 40, 43 -45). Some studies have required presence of an
adherent retroplacental clot or placental histopathology findings in addition to other signs
and symptoms(39, 41, 42, 45). At the extreme, a few studies have required cases to cross
a certain threshold of severity as evidenced by grade(46-48), necessity of immediate
delivery(49), or fetal demise(50). Information on diagnoses from birth certificates or
hospital discharge cOdes may not be uniform, both because practitioners may differ in
their propensity to apply the diagnostic label and because women with other high-risk
conditions or complications may receive more careful chart review and be more likely to
have abruptions documented in administrative records(51). Thus, although they have the
advantage of large samples, studies of abruption risk factors based purely on

administrative or vital data could perpetuate inflated estimates of the risks associated with

 

Table 1.1. Placental abruption case definitions from selected epidemiologic

studies

 

Author, Date

Williams 1992(52)
Bartha 1996(53)

Ananth 2001(35)
Ananth 2006(24)

Nurk 2004(36)

Casey 2005(54)
Ananth 1996(34)

Ananth 2005(27)
El Kady 2004(55)

Misra 1999(56)
Prochazka 2007(57)

Ananth 2007(38)
Nath 2007(44)

Kramer 1997(40)

Mousa 2000(58)
Hira 2002(48)
Agorastos 2002(46)
Anteby 2004(47)

’ Parle~McDermott
2005(59)

Naidu 2007(50)

Karakantza 2008(37)

Jaaskelainen 2008(39)

Larciprete 2007(41)

Placental abruption case definition
Not specified

Diagnosis entered on birth certificate

Birth registry

Perinatal database (hospital or regional)
Hospital discharge codes (lCD-9 or ICD-lO)
Clinical diagnosis (usually by attending physician at delivery)

Clinical diagnosis based on signs and symptoms (painful vaginal bleeding,
uterine pain, tenderness, hypertonicity); and/or retroplacental clot; and/or
prenatal sonographic diagnosis; confirmed by chart review

At least two of the following: 1) antepartum hemorrhage after 20 weeks’
gestation, 2) uterine pain or tenderness, 3) fetal distress or death, and 4)
retroplacental clot.

Placental abruption requiring immediate delivery

Abruption, grade 2 or 3

“Severe” placental abruption; defined as retroplacental clot and/or accidental
hemorrhage with associated clinical signs of abruption and/or a statement in the
case records that the patient was a definite case of abruption placentae.

Severe placental abruption resulting in stillbirth

Documented by routine second or third trimester ultrasound or in ultrasound
performed in response to bleeding and/or abdominal pain

Clinical diagnosis plus retroplacental clot, vaginal bleeding, uterine tenderness,
increased baseline tone by external monitor, or fetal distress or death

Clinical diagnosis based on antepartum uterine tenderness and vaginal bleeding
and confirmed by inspection of the placenta at delivery

 

 

Table 1.1 (cont’d).

 

Dizon-Townsend Clinical suspicion supported by written documentation (excessive bleeding,
2005(60) treatment with blood products, description of delivery and placenta, and/or
confirmation by pathologic exam

Wiener-Megnani Profuse vaginal bleeding not from placenta previa appearing during the third
1998(42) trimester of pregnancy and the clinical observation of the placenta
Zhang 2007(45) Clinical findings of vaginal bleeding without placenta previa, abdominal pain,

uterine tenderness, contractions; also placental blood clots compressing adjacent
villi

Salafia 1995(61) Clinical: antepartum vaginal bleeding judged clinically to be the primary
complication leading to preterm delivery
Pathological: gross retroplacental hematoma with subjacent placental infarct or
villous infarct with microscopic evidence of basal plate destruction or
deformation

McElrath 2008(8) Presentation with a significant amount of vaginal bleeding (documented in
medical record or postpartum hematocrit <24%) and a clinical diagnosis of
placental abruption in the absence of cervical change, among deliveries <28
weeks

 

well-known risk factors; on the other hand, such studies could underestimate risks
associated with less publicized or poorly documented risk factors.

Several lines of evidence implicate placental hemorrhage in the pathophysiology
of some preterm deliveries (PTD). Diagnosis of placental abruption is more commonly
made at preterm than term gestations(24). Early pregnancy vaginal bleeding has been
associated with both preterm delivery (PTD)(20, 21, 62) and placental abruption.(29, 31)
Hemosiderin ——suggesting old intrauterine bleeding — was found more often among very
preterm (<32 weeks) placentas than term placentas in one study(33). Intrauterine
(subchorionic or retroplacental) hematomas identified in early pregnancy by ultrasound
scans also marked women at high risk of preterm delivery and placental abruption(63).

Elevated maternal serum alpha-fetoprotein (MSAFP), a possible biomarker of disturbed

uteroplacental vascular integrity, has been consistently associated with increased PTD
risk(52, 64-68) and more strongly associated with abruption risk(52, 66, 68-72)

We hypothesized that clinically diagnosed placental abruption may be the tip of
an epidemiologic iceberg of placental hemorrhage. Clinically evident placental abruption
may only represent a portion of the clinically relevant involvement of bleeding in
delivery timing, because subclinical hemorrhage may also contribute to risk of PTD
without reaching a threshold necessary for clinical detection. Clinical diagnosis of
placental abruption is likely to be related to severity of signs and symptoms, presence of
risk factors, and intensity of prenatal care. Subclinical hemorrhage that predisposes a
woman to PTD could plausibly share similar risk factors with clinically diagnosed

abruption.

1.3. Polymorphisms in genes involved in vascular function in relation to evidence of
placental hemorrhage and preterm delivery

Because placental abruption involves an acute vascular disruption and has been
consistently linked to high blood pressure(73-76), underlying vascular causes have been
investigated in its etiology. At least two pathophysiologic mechanisms originating with
vascular dysfunction have been implicated in placental abruption risk. Normal pregnancy
alters hemostatis, shifting the balance to a relatively hypercoagulable state(77). Inherited
thrombophilias may exacerbate this shift and predispose women to develop decidual
artery thrombosis, which may lead to necrosis and venous hemorrhage, ultimately
resulting in a retroplacental hematoma(78). The renin-angiotensin- system regulates fluid

balance and blood pressure. An increase in angiotensin may result in blood vessel

constriction and sodium and water retention, thereby causing blood pressure to
increase,(79-8 l) and increased blood pressure may contribute to blood vessel rupture in
the matemal-fetal interface.

Recurrence risk for placental abruption is very high (~10-15%, reviewed by
Ananth, Savitz and Williams(73)), suggesting that factors specific to the mother —
perhaps genetic factors — may contribute to abruption risk. Candidate genes in vascular
pathways, including those involved in hemostasis and hemodynamics, have been
identified. Polymorphisms that have been implicated in a thrombophilic predispoSition
include methylenetetrahydrofolate reductase (M THFR) C677T, MTHF R A1298C, Factor
2 (prothrombin) G20210A, and Factor V (F5) G1691A (i.e. the Leiden variant,
abbreviated FVL). The C704T promoter polymorphism (also known as Met235Thr) in
the angiotensinogen (A G 7) gene, which codes for an angiotensin precursor peptide, has
been associated with increased risk of hypertension in some studies, although results have
been inconsistent(82).

Table 1.2 shows studies reporting on associations between selected
polymorphisms in M T HFR, F5, and AGT and placental abruption, adapted from a recent
HuGE review and metaanalysis by Zdoukoupoulos and Zintzaras(83). For MTHF R
polymorphisms, the metaanalysis provided little evidence that dominant models for
MTHFR(677) (based on 9 studies, pooled fixed effects OR=1.24 (95% CI 0.83, 1.85)) or
MTHFR(1298) (based on 3 studies, pooled fixed effects OR=1.33 (95% €10.97, 1.83))
conferred increased risk of abruption. For FVL, the metaanalysis identified significantly
increased abruption risk based on 10 studies, with a pooled random effects odds ratio of

3.42 (95% CI 1.62, 3.41). For the AGT C704T variant, only two studies of were included

in the meta-analysis; the larger (62 cases, 240 controls) found increased abruption risk for
the dominant model (OR=4.35), while the smaller (50 cases, 50 controls) found a non-
significant OR of 0.5.

Several published studies describing associations between these four
polymorphisms and placental abruption were not included in the Zdoukopoulos and
Zintzaras review and metaanalysis(36, 37, 41, 57, 84, 85) (see Table 1.2). Although no
table of excluded studies was presented, reasons that studies were not included in the
meta-analysis may be that they were been published too 1ate(57), were cohort (rather than
case-control) studies(36, 37, 84), were not published in English(85), and had incomplete
descriptions of genotype frequencies(41)). Results for the F VL dominant models from the
excluded studies that reported odds ratios were generally consistent with the studies
included in the meta-analysis (OR range 3.0 to 9.1)(3 7, 57, 84). The excluded studies
reporting on the two MTHF R polymorphisms did not report results of a dominant
model(36, 37, 41, 57).

The Zdoukopoulos and Zintzaras metaanalysis did not report pooled results for
recessive models. However, for the two MTHF R variants, the dominant model may not
be the biologically relevant model. Among ten studies that reported an odds ratio for the
MTHFR(677) TT allele —- either versus CT/TT or versus CC —— results have been
inconsistent, having odds ratios ranging from 0.58 to 2.65(36-38, 41, 50, 57, 59, 86-88).
Among five studies that reported an odds ratio for the MTHFR(1298) CC genotype, odds

ratios have ranged from 0 to 4.36(36, 38, 41, 59, 89).

10

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Fewer studies have been published on the associations between vascular function
genotypes and preterm delivery, either overall or by subtypes. Valdez-Velazquez found a
two-fold increased risk of preterm premature rupture of membranes with the homozygous
or heterozygous methionine genotypes (normally considered the major allele) vs. the
homozygous threonine genotype of the AGT polymorphism in Mexican women
compared with normal pregnancy community controls(95). Four studies specifically
reported on associations between FVL and preterm delivery, and none found an
association(84, 96-98). Of five studies identified that reported on associations between
MTHFR(677) and PTD(96-100), two found non-significant elevated risk with genotypes
with at least one copy of the T allele(98, 100). Finally, one study reported a null result for
MTHFR(1298) and PTD(96).

Few prior studies have studied genetic polymorphisms with respect to specific
placental lesions. Ariel and colleagues examined fetal and maternal vascular lesions in
relation to fetal inherited thrombophilias (FVL, prothrombin, or homozygous MTHFR
polymorphisms) in a series of 64 placentas from pregnancies complicated by
preeclampsia, placental abruption, or intrauterine growth restriction, and found no
association(lOl). Many and colleagues investigated 68 placentas from complicated
singleton pregnancies (i.e. those with severe preeclampsia, intrauterine growth
retardation, stillbirth or placental abruption). They compared 12 placental findings
between the 32 from women with at least one thrombophilia (FVL, MTHFR, or
prothrombin mutation or deficiency in proteins S, C, or antithrombin III) and the 36
having no thrombophilia(102). Only four placental findings differed significantly

between the thrombophilia and non-thrombophilia groups: the thrombophilia group had

19

lower placental weight, more frequently had single or multiple villous infarcts, and more
often had fibrinoid necrosis. Mousa et. al. used a similar design to study placentas from
79 pregnancies complicated by severe pre-eclampsia/eclampsia, placental abruption,
intrauterine growth restriction, or stillbirth; 43 women had at least one thrombophilic
abnormality (antithrombin III, protein ‘C, protein S, activated protein C resistance,
anticardiolipin antibodies, lupus anticoagulant, fasting plasma homocysteine and specific
mutations to methylenetetrahydrofolate reductase C677T, G20210A prothrombin gene
and factor V Leiden)(58). The majority of women in both groups had abnormal
histopathology, but no specific lesion pattern correlated with thrombophilia. These
studies have suffered from two important limitations. First, these studies considered
several inherited and acquired thrombophilias as a group even though their effects may
be heterogeneous. Second, none of these studies included normal pregnancy controls.
There may be multiple exposures (known thrombophilias, as-yet-identified
thrombophilias, or other abnormalities) that cause the placental abnormalities evident
among placentas from pregnancies having severe complications, and it may be
informative to compare thrombophilia status between pregnancies with complications to

those with normal outcomes.

1.4. Evidence of placental hemorrhage in relation to inflammation and infection
It is well-accepted from many lines of evidence that ascending infection is

causally related to some preterm deliveries, especially early preterm deliveries(l 1).

Subclinical infections in gestational tissues are believed to trigger an inflammatory

cytokine cascade that causes uterine contractions resulting in either spontaneous preterm

20

labor (SPTL) or membrane degradation resulting in premature rupture of membranes
(PROM). Another pathway to spontaneous preterm delivery, decidual hemorrhage, has
been described by Lockwood(103). This “bleeding pathway” is sometimes conflated with
the clinical circumstance of placental abruption(104); however it may be one mechanism
of several leading to clinically diagnosed placental abruption. Limited epidemiologic
evidence has linked placental abruption, decidual hemorrhage, or other gestational uterine
bleeding to infection or other inflammatory processes. Studies that have investigated
associations between evidence of placental hemorrhage and evidence of intrauterine
infection are summarized in Table 1.3.

An analysis of data from the National Maternal and Infant Health Survey found
that, at preterm gestations, abruption diagnoses are associated with diagnoses of acute
inflammation-related conditions such as preterm PROM and intrauterine infections(24).
Several other articles have reported statistical associations between diagnosed
chorioamnionitis or intrapartum fever and abruption(27, 29, 105). Moreover, abruption is
recognized (by the American College of Obstetricians and Gynecologists among others)
as a possible complication of preterm PROM(73, 105-107). If the PROM has an
inflammation-related etiology (e.g., Ananth et. al. grouped PROM with non-PROM
intrauterine infections as “acute inflammation”—associated conditions(24)), then any
resulting abruption could be indirectly attributable to inflammation (perhaps via
subsequent reduced uterine volume and attachment surface area(105)) or more directly
attributable if the inflammatory process causes placental vessel friability resulting in

decidual hemorrhage(1 08).

21

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27

A handful of studies have explored a possible connection between hemorrhage
and infection using placental pathology examinations, with contradictory findings. In the
early 19803, using data from the National Collaborative Perinatal Project (N CPP), Naeye
found an association between “deciduo-chorio—amnionitis” and placental abruption(112).
A more recent report using NCCP data reported modest (RR range 1.2 -— 1.8) associations
between placental lesions indicative of acute or chronic inflammation and placental
abruption(3l). A case-control study conducted in South Africa examined placentas from
30 cases of severe placental abruption and 60 non-abruption gestational age-matched
controls, and found no association with measures of fetal or maternal inflammatory
response(1 13). A single-hospital cohort study representing 36,875 births from 1978 to
1989 had diagnostic placental examination data on all deliveries. The authors found an
association between placental abruption and histologic chorioamnionitis, with an odds
ratio of 2.50 (95% CI 1.58, 3.98) after adjusting for maternal age, marital status,
smoking, pre-pregnancy hypertension, pregnancy-induced hypertension, prolonged
rupture of membranes, fetal sex, and small for gestational age fetus.(40) Recently, in the
New Jersey Placental Abruption Study, a case-control study of abruption etiology, Nath
et. al. found that severe HCA (defined by the authors as >30 neutrophils per high power
field) was more common among abruption cases than controls; this pattern was similar
for term and preterm deliveries(44).

In a series of placentas delivered prior to 32 weeks, Salafia found that
hemosiderin deposition, indicative of previous hemorrhage, was inversely associated with
histologic evidence of acute ascending infection but positively associated with chronic

inflammation in the basal plate(33). Using another gestational age-truncated sample,

28

McElrath et. al. performed a factor analysis of a wide range of dichotomized' variables
(i.e. maternal demographics, newborn characteristics, placental histopathology, and
placental microbiology) using data from the Extremely Low Gestational Age Newborn
(ELGAN) Study(8). From this analysis, which included only births occurring prior to 28
completed weeks gestation, the authors concluded that placental abruption belonged with
preterm labor, preterm premature rupture of membranes, and cervical insufficiency in an
infection/inflammation group, while preeclampsia and preterm birth for fetal indication
belonged together in a group characterized by impaired placentation.

Although placenta is an important tissue to study for evidence of infection, some
studies have linked infection and bleeding using other biologic samples. Much of the
research relating infection to preterm birth has focused on microbes ascending from the
vaginal tract. Another area of inquiry has related to observed associations between
periodontal disease/oral pathogens and risk of preterm birth (recently reviewed by
Boggess et. al (116)). One study explored the association between antepartum vaginal
bleeding, umbilical cord serum IgM-positive for oral pathogens, and risk of preterm birth
at less than 35 weeks(110). The authors found evidence for effect modification, in that
pregnancies with antenatal vaginal bleeding and fetal exposure to oral pathogens had a
much higher risk of preterm birth than those with either exposure alone. They speculated
on two potential mechanisms to explain their findings. First, the bleeding may have been
evidence of a “disruption of the matemal-fetal interface” that allowed oral pathogens
already present in the maternal circulation into fetal contact, which initiated a fetal

response leading to PTD. Second, a fetal inflammatory response to oral pathogens from

29

another exposure route may have caused decidual inflammation that led to vaginal
bleeding.

A prospective cohort study found that first trimester bleeding was associated with
T richomonas vaginalis (TV), Chlamydia trachomatis, and bacterial vaginosis (BV);
further, that study found that women with both bleeding and BV or TV had a higher risk
of spontaneous preterm birth than women with bleeding or infection alone(108). The
authors suggested two possible mechanistic reasons for this apparent interaction: either
the vaginal infection progresses to deciduitis/endometritis and causes clinically
recognized bleeding, or bleeding from the decidua or trophoblast facilitates microbial
ascent, interferes with host defenses, and/or serves as a substrate for bacteria, supporting
the advancement of infection, and further aggravating the course of pregnancy.

A study from the Perinatology Research Branch at NICHD approached the
infection-bleeding link by studying amniotic fluid from 114 women who had idiopathic
vaginal bleeding during pregnancy( 1 17). The bleeding was confirmed to be of uterine
origin, and not attributable to placenta previa or overt placental abruption. Sixteen (14%)
of these women had positive amniotic fluid bacterial cultures; this subset had the worst
outcomes. The women with positive cultures were more likely than those with negative
cultures to have histologic chorioamnionitis, subsequent preterm premature rupture of
membranes, and spontaneous PTD, with 77% delivering prior to 28 weeks compared with
27.5% of the women with bleeding but negative cultures. Importantly, the study did not
include a comparison group of non-bleeding women. The authors concluded that vaginal
bleeding in these cases was a symptom of an otherwise subclinical amniotic fluid

infection; however, they did not consider alternative explanations, such as that the

30

bleeding preceded and provided a nutritive substrate for bacteria, or that bleeding was
unrelated to the infection. I

In evaluating the strength of evidence from studies attempting to link the bleeding
and infection/inflammation pathways to preterm birth, careful consideration should be
given to several methodologic issues. Studies from large administrative databases
typically have adequate sample sizes of abruption cases; however they can provide no
information on time-order, biomarkers, or pathology, and inferring mechanisms from
such studies is difficult. Further, since all information on outcomes and most information
on putative exposures are derived from the same administrative data sources, non-
differential misclassification and shared method variance may result in biased estimates
of the degree of association, therefore these studies can not be considered conclusive with
respect to determining any causal link between infection and abruption.
For inferring presence of intrauterine infection, studies have used either clinical(24, 29,
105, 108) or histologic(3l, 40, 44, 109, 112-115) determinations, which might result in
different conclusions. Placental examination is the most objective way to quantify both
infection and hemorrhage. Unfortunately, it is not possible to definitively establish time
order after delivery has occurred. Moreover, studies differ in their placental pathology
measures, with some using clinical — rather than research protocol — placental exams, and
the pathologist’s knowledge of the clinical picture may have influenced their diagnoses. ‘
Even among studies that have employed research protocols, criteria used to define
histologic chorioamnionitis have varied considerably, as recently reviewed in the context
of preterm delivery.(19) Many of the studies reviewed here have been limited by not

having a comparison group of term pregnancies (or placentas) or pregnancies

31

uncomplicated by abruption or bleeding.(8, 44, 61, 115) This is important because both
bleeding and infection are causes of preterm delivery, thus in this instance preterm
delivery may be a collider variable - that is, a common effect of the two variables of
interest.(l 18) Investigating the association between bleeding and infection only among
preterm deliveries is a form of collider stratification, which can induce spurious

associations between variables.

1.5. Aims

The aims of this dissertation are:

1. To evaluate four indicators of placental hemorrhage (first trimester bleeding,
placental abruption, disc-impacting blood clots, and high scores on 3 Microscopic
Vascular —— Disturbance of Integrity construct) as potential components of a
common “bleeding pathway” by (1) assessing their mutual associations, (2)
describing their prevalence according to maternal characteristics, and (3)

estimating their odds of preterm delivery and its subtypes;

2. To assess the associations between four polymorphisms in three candidate
maternal genes in thrombophilia and renin-angiotensin system pathways and

preterm deliveries subtypes defined by evidence of placental hemorrhage;

3. To compare histologic chorioamnionitis with clinical chorioamnionitis, and to
evaluate risk of histologic chorioamnionitis and clinical chorioamnionitis in

relation to evidence of placental hemorrhage ascertained at different times in

pregnancy.

32

CHAPTER 2.

EVIDENCE OF PLACENTAL HEMORRHAGE AND PRETERM DELIVERY

2.1. Introduction

Several etiologic pathways have been implicated in preterm delivery (PTD),
including infection, stress, abnormal uterine distension, and uterine bleeding.(3) Various
types of direct or indirect evidence of placental hemorrhage (PH) may be manifestations
of a uterine bleeding pathway. Vaginal bleeding in early to mid-pregnancy has been
associated with PTD in many studies.(20, 21 , 62) Placental abruption, or premature
separation of a normally placed placenta, is a potentially severe pregnancy complication
that may originate with bleeding into the decidua from a ruptured arteriole(119) and can
result in major obstetric hemorrhage. Placental abruption is present in a larger proportion
of preterm than term deliveries.(24, 29) Subchorionic or retroplacental bleeding detected
by prenatal ultrasonography(63, 120) and hemosiderin (a breakdown product of old
blood) in placental tissues detected through histologic examination(6l) have also been
associated with PTD, although these have not been reported on extensively.

It is unclear whether these variable manifestations of bleeding belong to a single
pathway with a common set of maternal risk factors. Vaginal bleeding early in pregnancy
has been linked to placental abruption(20, 31, 62), suggesting that bleeding and abruption
-- both clinically evident indicators of PH -- may share common pathways. Many other
maternal characteristics and clinical circumstances are shared risk factors for both PTD
and placental abruption, including hypertension(121-123), elevated maternal serum

alpha-fetoprotein (MSAFP)(52, 66, 68), Afi'ican-American identity(124, 125),

33

smoking(122, 126), cocaine abuse(127), and low body mass index (BMI)(128, 129). Less
has been published on maternal characteristics associated with other evidence of PH,
such as early pregnancy bleeding(130) or placental pathology findings.

While placental abruption is rare, complicating approximately 1% of
pregnancies(28), placental pathology findings consistent with abruption are more
prevalent(1 31). We hypothesized that placental abruption may only represent a portion of
the uterine bleeding that affects delivery timing — the “tip of the iceberg” — because
subclinical gross or microscopic evidence of placental hemorrhage apparent only in
placental examinations might also be related to PTD risk. The prospective Pregnancy
Outcomes and Community Health (POUCH) Study collected multiple sources of
evidence of PH, including mid-pregnancy maternal reports of prior vaginal bleeding
episodes, clinical data from medical records and gross and microscopic placental
pathology findings. The aim of this study was to evaluate four indicators of PH as
potential components of a common “bleeding pathway” by (1) assessing their mutual
associations, (2) describing their prevalence according to maternal characteristics, and (3)

estimating their odds of PTD and its subtypes.

2.2. Methods
Study Protocol

The POUCH Study enrolled 3019 pregnant women from five Michigan
communities at 15-27 weeks’ gestation. English-speaking women aged 215 years who
had MSAF P screening at 15-22 weeks, a singleton pregnancy with no known congenital

anomalies, and no pre-existing diabetes were eligible. MSAFP was of particular interest

34

in the POUCH Study’s design because of this biomarker’s consistent association with
PTD(6); thus, the POUCH cohort oversampled women who had unexplained high
NSAF P (32 multiples of the median, 7% of cohort). The POUCH study protocol
received institutional review board approval from Michigan State University, the

Michigan Department of Community Health, and all nine delivery hospitals. All women

provided informed written consent.

At enrollment, women participated in a structured interview with a study nurse
and completed a self-administered questionnaire. The interview elicited information on
demographics (including self-reported race/ethnicity), height, pre-pregnancy weight,
reproductive history, medical conditions and events during pregnancy. During the

interview, women were asked, “Have you had any spotting or bleeding so far during this
pregnancy?” and prompted to describe timing (gestational week) and heaviness (spotting,
Slight, about the same as usual period, or heavier than usual period) for up to 7 episodes.
The questionnaire was designed to collect data on potentially sensitive questions
including substance use and physical abuse. Women were asked how often in the last 6
months they had been “shoved, hit, or physically abused by [their] parents or a partner or

husband” (never, once or twice, several times, often, or very often).

Gestational age was estimated by last menstrual period (LMP), corroborated by an

ultrasound (US) scan conducted prior to 25 weeks (available for 97% of women). If the
e stimates differed by more than 2 weeks (17%) or if LMP was unavailable (3.6%), the

ultrasound date was used. PTD was defined as delivery prior to 37 completed weeks’

gestation.

35

Subcohort Sample
.A subcohort (n=1 371) was selected for detailed study of biologic samples and
chart-level data. The subcohort included all PTDs, all women with unexplained high
IvISAFP and a stratified sample of women with normal MSAF P and term deliveries, with
oversampling of African-Americans. Subcohort analyses incorporate sampling weights to
reconstitute the cohort distributions and further correct for overrepresentation of high
:MSAF P in the cohort, such that weighted proportions based on the subcohort can be
interpreted as prevalence or risk.
J’lacenta protocol
After subcohort deliveries, placentas were formalin fixed at the delivery hospital
prior to transport to the study’s pathology laboratory. For the gross examination, parallel
slices were made through the placental disc 1 cm apart. The pathologist noted clots in the
cut surface, along with indicators of adjacent tissue involvement, i.e. dissecting
hemorrhage, and tissue infarction, compression, or red/brown discoloration. Disc-
impacting blood clot was defined as a gross examination finding of a retro- or
intraplacental blood clot impacting adjacent tissue.
For the microscopic placental examination, nine tissue samples per placenta were
8 elected: two from the membrane roll, two from the cord, and five fi'om the disc(19).
Microscopic vascular-related findings that fell within five constructs adapted from a
d iagnostic coding tool were recorded. Items in the “Maternal Vascular - Disturbance of
I mtegrity” (MV—I) construct, i.e. microscopic evidence of retroplacental blood with
adj acent disc disruption/compression, decidual hemorrhage in the basal plate, and

Qecidual hemosiderin-like pigment in the membranes or basal plate, were used to

36

calculate an MV-I score for each woman. Microscopic hemorrhage was defined as the
top quintile of MV-I scores based on the distribution of scores among term deliveries
with normal MSAF P. This serves as a possible indicator of atypical maternal vessel

hemorrhage. This distributional cut-point along a continuum of findings was previously

shown to correlate with PTD risk(23).

.Medical record abstraction

Study nurses abstracted subcohort medical records in detail. For all PTDs, an
additional brief abstraction was completed by a physician with expertise in obstetrics. A
pool of possible placental abruption cases, identified by bleeding near delivery or any
mention of placental abruption in the abstracted data, were later reviewed by three
clinicians with labor and delivery experience who were unaware of the placental
pathology findings recorded by the POUCH Study pathologist (PKS). Placental abruption
was defined as (l) documented signs and symptoms consistent with abruption (e. g.
vaginal bleeding, pain, increased uterine tone, fetal distress); or (2) retroplacental

hematoma visualized on a prenatal ultrasound scan. Disagreements among the reviewers
Were resolved by discussion with the principal investigator (CBH) and study pathologist
( PKS) until a consensus was reached.

Other relevant information abstracted from patient charts included trauma or
injuries during pregnancy, episodes of vaginal bleeding during pregnancy and date of
Occurrence, blood pressure and proteinuria values, and details of the delivery process
i licluding timing of membrane rupture, cervical dilatation, and interventions.
Iflypertension was defined as documented diastolic blood pressure 290 and/or systolic

blood pressure 2140 on 22 days or a documented diagnosis and/or history of

37

hypertension, or anti-hypertensive medication prior to 20 weeks. Preeclampsia,
gestational hypertension and chronic hypertension were not considered separately
because numbers of each were small. PTD subtypes, including medically indicated,
spontaneous labor, and premature rupture of membranes (PPROM) were determined
based on chart-level data as previously described(14). Spontaneous PTD included
spontaneous preterm labor and PPROM.
Evidence of placental hemorrhage
The four principal PH indicators compared in this study were (1) placental
abruption, (2) disc-impacting blood clots, (3) microscopic hemorrhage, and (4) first
trimester bleeding (any vs. none, ascertained during enrollment interview). Two
additional variables describing vaginal bleeding during pregnancy were used instead of
first trimester bleeding (any vs. none) in selected analyses: (1) first-trimester bleeding
from the enrollment interview categorized as heavier than spotting, spotting only or none,
and (2) first and second trimester vaginal bleeding (excluding bleeding in the week
before delivery) recorded in prenatal charts categorized as first trimester only, second
1fl‘irnester only, both trimesters, or none.
Study Sample
From the subcohort, we sequentially excluded 158 women whose placentas were
not saved, 127 women whose placental exams were not yet complete, 6 women with
131 acenta previa at delivery, and 84 women who identified themselves as any race/ethnic
c‘c‘ltegory other than “White or Caucasian” (referred to as “white”) or “Black or Afiican-

American” (referred to as “Afiican—American”). After these exclusions, a sample of 996

U V Omen remained.

38

Descriptive statistics

As a first step, we calculated the prevalence of the four PH indicators and
maternal characteristics overall and stratified by delivery timing (term/preterm). Next, we
calculated the prevalence of each PH indicator among women with each of the other three
indicators, and calculated bivariate odds ratios (OR) and 95% confidence intervals (CI)

for each pair of PH indicators. Finally, we estimated the prevalence of each indicator

according to selected maternal characteristics, and tested for differences in proportions

msing modified Rao-Scott )6 tests for complex survey designs(1 32).

Modeling strategy

We used weighted logistic regression models to assess relations between the four
PH indicators and PTD. We calculated ORs for each PH indicator individually, then
entered all four variables into the same model (i.e., a “mutually adjusted” model). Next,
we added maternal characteristics to the model, and retained those that changed ORs for
any of the PH indicators by 25% from the mutually adjusted estimates. Potential
confounders considered were those associated with PTD or any of the PH indicators in
uni variate analyses. Finally, we added hypertension separately to examine its effect on
O‘Il'ler variables in the model. Hypertension may have a direct, proximate effect on

placental abruption, placental pathology findings, and delivery timing.

Weighted polytomous logistic regression models were used to assess the relations
betWeen PH indicators and PTD subtypes defined by timing (35-36 weeks, <35 weeks,

terlrr [referent]) or clinical circumstances (spontaneous, medically indicated, term

[referent]; and spontaneous labor, PROM, medically indicated, term [referent]).

39

All statistical analyses were conducted using SAS 9.1.3 (Statistical Analysis

Software, Cary, NC).

2.3. Results

Descriptive statistics

Characteristics of the subcohort sample are detailed in Table 2.1, overall and
stratified by delivery timing. In weighted analyses, 10.7% of pregnancies ended in PTD.
In the total sample, prevalence of the four PH indicators ranged from 2.0% for placental
abruption and 5.6% for disc-impacting blood clots to about 20% for microscopic
hemorrhage and first trimester bleeding. All four PH indicators were more prevalent
among PTDs than term deliveries. Medicaid insurance, lack of high school education,
spending > 1 hour per week in a smoky room, hypertension and parity/prior PTD were

also associated with PTD in these univariate analyses.

Table 2.2 shows pairwise associations among the four PH indicators. Although
placental abruption was associated with all three other PH indicators (versus disc-
impacting blood clot, OR=5.5 (1.7, 17.3); versus microscopic hemorrhage, OR=2.3 (1.0,
5 . 5); versus first trimester bleeding, OR=3.4 (1.4, 8.5), less than 50% of placental
abruption cases had each of these other PH indicators (23.0%, 37.0%, and 43.8%,
respectively). Disc-impacting blood clot and microscopic hemorrhage were strongly
as sociated with one another (OR=4.6 (2.3, 8.9)), but first trimester bleeding was not
associated with disc-impacting blood clot or microscopic hemorrhage (0R3 1.1 and 0.9).
Figure 2.1 shows the number of women with all combinations of the four PH

i l'ldicators. A total of 4] 3 women (38.9% weighted) had at least one PH indicator, and

40

321 women (31.9% weighted) had only one PH indicator. Nine of 31 placental abruption
cases (35.2% weighted) had no other PH indicators.
Maternal characteristics had few statistically significant associations with
evidence of PH (Table 2.3). Women with <12 years of education had a lower prevalence
of first trimester bleeding than women with more education. Women with high MSAF P
had a higher prevalence of both placental abruption and first trimester bleeding than
women with normal MSAF P. Smokers had a lower prevalence of microscopic
hemorrhage than non-smokers. Hypertensive women had a higher prevalence of disc-
impacting blood clots than non-hypertensive women. Nulliparous women had a lower
prevalence of all four PH indicators than parous women with and without prior PTD,
although only the association with placental abruption was statistically significant.
Model Results
For PTD <37 weeks, there were elevated odds for each PH indicator in unadjusted
analyses (Table 2.4). In a mutually adjusted model (i.e., a model that included all four PH
indicators), all four ORs were attenuated, and disc-impacting blood clot lost statistical
Significance. Only minor changes occurred afier adding maternal characteristics to the
rITIodel. Adding hypertension to the model slightly attenuated the ORs for placental
abruption and disc-impacting blood clot but had no impact on the other two PH
indicators. When we considered two alternative specifications of vaginal bleeding during
pregnancy in these models, we found that heavier bleeding had a stronger association
V"ith PTD than spotting, and bleeding in both first and second trimesters (from patient
cl"larts) had a stronger association with PTD than bleeding confined to either the first or

SeCond trimester in unadjusted but not adjusted analyses (not shown). However, neither

41

of these alternative specifications changed the interpretations for the other three PH
indicators, thus all further analyses use first trimester bleeding (any vs. none) from the
maternal interviews.

When we stratified PTD by gestational week, all four PH indicators had stronger
associations with PTDs at <35 weeks than with PTDs at 35-36 weeks in all models
(Table 2.4). All four PH indicators were significantly associated with PTD <35 weeks in
all models.

When we stratified PTD by delivery circumstances, ORs did not differ
meaningfirlly between spontaneous and medically indicated PTDs in unadjusted,
mutually adjusted, or maternal characteristics-adjusted models (Table 2.5). After adding
hypertension, the OR of 2.2 for disc-impacting blood clot and medically indicated PTD
observed in the maternal characteristics-adjusted model was reduced to 1.1. Although
numbers were very small in some cells, we further subdivided spontaneous PTDs into
spontaneous labor and PPROM. Only microscopic hemorrhage was associated with
PPROM (adjusted OR = 2.0, 95% CI 1.1, 3.5); the other three measures had stronger

associations with spontaneous labor (not shown).

42

 

Table 2.1. Indicators of placental hemorrhage, maternal characteristics and risk
factors, overall and by preterm delivery status

 

 

 

Total Term Preterm P 1'
N %* N "/o* N °/o*
Total 996 758 89.3 238 10.7
Indicators of Placental hemorrhage
(not mutually exclusive)
Placental abruption 31 2.0 12 1.4 17 7.3 <.0001
Cut surface clot impacting adjacent 62 5.6 36 5.0 26 10.4 .005
tissue
Microscopic hemorrhage 215 20.4 143 19.3 72 29.4 .002
First Trimester Vaginal Bleeding 220 19.2 154 18.2 66 27.8 .005
Maternal characteristics
African-American 416 24.6 340 23.6 76 32.9 I
Medicaid-insured 5 36 45 .6 41 1 44.8 125 52.7 .03
Education: did not complete high 203 16.3 153 15.7 50 21.6 .03
school
High MSAFP 159 3.5 128 3.1 31 7.1 I
Smoked during pregnancy 282 26.8 209 26.3 73 30.6 .23
Smoked prior to pregnancy 343 33.1 250 32.4 93 38.5 .10
Smoky room > 1 hr/wk 479 44.9 349 43.6 130 55.6 .002
Cocaine use (ever) , 82 8.5 25 10.7 57 8.3 .29
Trauma/injury noted in medical record 116 10.7 89 10.6 27 11.8 .62
Physical abuse (previous 6 months) 105 9.1 83 9.1 22 8.8 .89
Hypertensive disorder (includes 99 8.6 61 7.7 38 16.3 <.01
preeclampsia, pregnancy-induced
hypertension, and chronic
hypertension)
Age
<20 160 13.3 122 12.9 38 16.3 0.36
20-34 765 78.6 583 78.8 182 77.0
35+ 71 8.1 53 8.3 18 6.7
Parity/prior preterm birth
Nulliparous 407 40.8 304 40.5 103 43.6 <0.0001
Parous/no prior preterm birth 449 49.2 380 51.7 69 28.6
Prior preterm birth 139 9.9 74 7.8 65 27.7
Prepregnancy Body Mass Index
<18.5 44 3.6 30 3.3 14 6.1 0.13
18.5 - 24.9 438 45.4 336 45.8 102 42.5
25-29.9 228 24.7 179 25.1 49 21.3
230 286 26.3 213 25.8 73 30.1

\

 

*Percentages have been weighted (inverse of sampling probability) to reflect distribution in cohort.
TP-value for preterm vs. term comparison by Rao-Scott chi-square test (SAS PROC SURVEYFREQ).
IP-value can not be calculated in univariate analyses because exposure and outcome are sampling strata.

43

 

 

 

 

 

 

 

 

 

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44

 

Table 2.3. Prevalence (weighted row percent) of four indicators placental
hemorrhage according to selected maternal characteristics

 

Overall

Race/ethnicity
African-American
White

Medicaid-insured
Yes
No

Education
<1 2 years

212 years

MSAF P
Unexplained high
Not high

Smoked during pregnancy
Yes
No

Smoked 6 months before
pregnancy

Yes
No

Time spent in smoky room
21 hr/wk
<1 hr/wk

Ever used cocaine
Yes
No

Placental
abruption

2.0

3.2
1.7

2.2
1.9

2.9
1.9

5.7*
1.9

1.1
2.4

1.3
2.4

2.3
1.8

2.0 _

2.0

impacting
blood clot

45

5.6

5.9
5.4

5.7
5.4

6.0
5.5

7.8
5.5

4.6
5.9

4.5
6.1

5.5
5.6

4.7
5.6

Microscopic
hemorrhage

20.4

18.9
20.9

17.7
22.7

16.6
21.1

23.5
20.3

15.0”“
22.4

16.7
22.3

19.2
21.4

13.4
21.1

First
trimester
bleeding

19.2

18.2
19.5

16.9
21.1

13.4*
20.3

32.9*
18.7

15.8
20.5

15.5
21.0

19.2
19.2

16.1
19.5

 

Table 2.3 Leont’d)

 

Placental
abruption
Trauma! injury noted in
medial record
Yes 5.4
No 1.6
Physical abuse (previous 6
months)
Yes 1.5
No 2.1
Hypertensive disease
Yes 4.0
No 1.9
Age
<20 2.1
20-34 2.1
35+ 1.4
Parity/prior preterm birth
Nulliparous 0.8*
Parous/no prior
preterm birth 2,7
Prior preterm birth 3 .9
Prepregnancy Body Mass
Index
<18.5 7,1
18.5 - 24.9 2.1
25-29.9 1.7
230 1.5

Disc-

impacting

blood clot

3.6
5.8

8.1
5.3

15.3*
4.6

5.4
5.4
7.0

4.4

6.1
7.3

7.7
4.5
6.6
6.0

Microscopic
hemorrhage

17.5
20.8

23.0
20.1

24.3
20.0

20.7
20.3
20.7

18.5

20.6
27.3

16.3
21.7
20.7
18.5

First
trimester

bleeding

17.0
19.5

21.7
18.9

19.3
1.92

14.5
19.8
21.1

17.6

19.3
25.6

20.1
21.3
20.6
14.1

 

*P<.05 from Rao-Scott modified chi-square test.

46

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48

 

Fltst Trimester —.
Bleeding

Placental Abruption

  
  
  

1 61
Dlsc-lmpacthg Blood Clot

 

Mlcrosooplc

 

Figure 2.1. Venn diagram showing 15 possible combinations of the 4 indicators of
placental hemorrhage. Disc-impacting blood clot = oval, double line, microscopic
hemorrhage = rectangle, double line; first trimester bleeding = rectangle, single line;
placental abruption = oval, single line. Note: 583 women with none of the 4 types of

evidence are not represented.

49

2.4. Discussion

A key finding of this study was that evidence of PH identified through objective
gross and microscopic placental pathology exams (i.e. disc-impacting blood clot and
microscopic hemorrhage) were associated with PTD at <35 weeks even after accounting
for clinically evident bleeding (i.e. placental abruption and first trimester bleeding).
Previously, Salafia et. al. found that hemosiderin in the decidua or extraplacental
membranes was more common in very preterm (<32 weeks’ gestation) deliveries than in
a sample of uncomplicated, healthy term deliveries in an unadjusted analysis(61).
Associations between early pregnancy vaginal bleeding and subsequent PTD(20, 21, 62,
133) or placental abruption(29, 31, 62) have previously been identified. Our study builds
on these prior findings by considering these clinically evident manifestations of placental
hemorrhage in conjunction with subclinical placental pathology findings.
Prior studies have shown that pathology findings consistent with placental
abruption are more common than clinical diagnoses.(119, 134) Some have argued that
placental pathology-based abruption-related findings are inconsequential in the absence
of clinical suspicion, given that most pregnancies with such findings have unremarkable
outcomes(28). In the POUCH Study, two types of evidence of placental hemorrhage from
pathology exams — disc-impacting blood clot (prevalence 5.6%) and microscopic
hemorrhage (“prevalence” 20.4%, based on a distributional cut-point) — were clearly
more common than placental abruption cases (prevalence 2.0%), and the majority of
women with disc-impacting blood clots and high microscopic hemorrhage scores
delivered at term without placental abruption. However, after accounting for placental

abruption cases in multivariable models, we identified excess PTD risk associated with

50

both of these placental pathology findings, particularly for PTDs occurring at <35 weeks.
Few studies have empirically demonstrated an association between subclinical evidence
of hemorrhage in the delivered placenta and PTD by comparing preterm and term
placentas. In prior work from the POUCH Study(23), the microscopic hemorrhage
construct was found to be associated with both spontaneous and medically indicated
PTDs occurring at <35 weeks. However, information on prior vaginal bleeding episodes
and placental abruption were not considered in that study, thus it was unknown whether
the observed results were primarily attributable to clinically evident hemorrhage.

Since retroplacental clots are sometimes employed in placental abruption clinical
diagnoses, and have been required to confirm placental abruption cases in some
epidemiologic studies(3 9, 45, 112), the limited association between placental abruption
and disc-impacting blood clots is noteworthy. It is important to distinguish the disc-
irnpacting blood clot construct captured by the POUCH Study pathologist from adherent
retroplacental clots visualized on a freshly delivered placenta by an attending clinician.
Data on the latter is not uniformly recorded in patient charts, thus we relied on a gross
placental pathology protocol to infer presence of a clot prior to delivery. Collected blood
not resulting from hemorrhage can become sequestered in the space between the
membranes and disc and become firm with formalin fixation, resembling a true clot from
hemorrhage. Thus, the pathologist identified clots associated with disc tissue changes that
would support a significant retroplacental clot on gross examination alone. Disc-
impacting blood clots undiagnosed as placental abruption may signal concealed (possibly
intraplacental or dissecting) hemorrhage, a less severe clinical picture, or a case with a

low index of suspicion for placental abruption. Placental abruption diagnoses in the

51

absence of disc-impacting blood clots could occur in very acute abruptions in which clots
do not have time to organize and cause tissue reactions prior to delivery(135).

We found that microscopic hemorrhage was modestly associated with PTD
(unadjusted OR=1.7), and was minimally affected by adjustment for the other types of
evidence of placental hemorrhage, maternal demographic characteristics, or hypertension:
While it is possible that findings in this construct represent intermediate steps on a
pathway that sometimes leads to overt placental abruption, most cases of placental
abruption (63%, see Table 2) did not have high scores on this construct. We suspected
that gestational vaginal bleeding remote from delivery, which has been linked to
placental abruption and PTD in this and other studies(20, 21, 29, 31, 62, 136), might
signal chronic, slow hemorrhage, and would thus be associated with high microscopic
hemorrhage scores, but this was not the case. Microscopic hemorrhage was associated
with PPROM, while the three other manifestations of placental hemorrhage were not.
Possible mechanisms linking specific items included in our microscopic hemorrhage
construct to spontaneous PTD and PPROM have previously been suggested: thrombin
generated in response to decidual hemorrhage may cause cervical ripening, contractions,
and membrane degradation resulting in preterm labor or PPROM(103); and tissue
hemosiderin, an iron compound produced during the breakdown of blood, may exert
tissue irritant and pro-inflammatory effects that contribute to preterm 1abor(33).
Additional research is needed to discover the antecedents of high microscopic
hemorrhage scores.

The strengths and limitations of this study primarily derive from the prospective

cohort design and the placental pathology protocol. Statistical power to detect significant

52

53

F?-

In

associations differed among the four PH indicators given their widely varying prevalence,
which ranged from 2.0% to 20.4%. Sample size limitations precluded assessing risk of
very early PTDs (i.e. <32 weeks). However, performing the pathology exam on a large
sample of term and preterm deliveries allowed us to empirically evaluate the relations
between PTD and four PH indicators. Like most studies that have investigated gestational
vaginal bleeding in relation to pregnancy outcome, we cannot discern the actual origin of
the blood — some may be from the placenta, while other reported bleeding may have
originated in the cervix or vaginal tract. The study population was drawn from a well-
characterized cohort, which was very similar to community women giving birth in the
study years based on birth certificate comparisons.(137) Data on first trimester bleeding
and substance use were ascertained prospectively, therefore this information was not
subject to differential reporting based on mother’s or clinician’s knowledge of pregnancy
outcome. The subcohort sampling scheme is not a source of bias because appropriate
weighted analyses were performed.

Each of the four manifestations of PH considered in this study contributed
information regarding PTD risk. Some of the PH indicators were associated with one
another, but others were not. Many placental abruption cases did not show strong
evidence of PH in their placental pathology examinations. The PH indicators diverged
somewhat with respect to their relations with maternal characteristics and PTD subtypes.
Taken together, these findings suggest potential heterogeneity in the hypothesized PH
“iceberg”. In the future, it may be helpful to consider both clinical and subclinical

manifestations of PH in relation to biomarker data (e. g. gene polymorphisms, anti-

53

angiogenic factors) in order to gain insight into broader pathways to PTD that may

involve disrupted uteroplacental vascular integrity.

54

CHAPTER 3.
POLYMORPHISMS IN THROMBOPHILIA AND RENIN-ANGIOTENSIN

SYSTEM PATHWAYS, PRETERM DELIVERY AND EVIDENCE OF
PLACENTAL HEMORRHAGE

3.1. Introduction

Preterm delivery (PTD), defined as delivery prior to 37 completed weeks’
gestation, complicates more than 10% of pregnancies in the United States, and
contributes to the burden of neonatal morbidity and mortality.(3) Several pathways have
been implicated in the etiology of PTD, including infection, stress, uterine distension, and
uterine bleeding.(3) Placental abruption (PA), a rare pregnancy complication
characterized by premature placental detachment, is strongly associated with PTD.(138)
Moreover, other less severe concealed or subclinical placental hemorrhage evident only
in placental pathology examinations may mark excess PTD risk(33). Although it has not
been established whether all manifestations of placental hemorrhage belong to a common
disease pathway, we hypothesize that preterm PA cases are the “tip of an iceberg,” the
most extreme manifestations of a broader hemorrhage-related pathway.

Recurrence risk for PA is very high(56, 139), potentially suggesting a role for
genetic factors. Hemostatic and hemodynarnic dysregulation are two vascular
mechanisms that have been implicated in PA risk. Two polymorphisms in the
methylenetetrahydrofolate reductase (M T HFR) gene and one polymorphism in the Factor
V (F 5) gene (i.e. the Leiden variant, F VL) have been linked to a thrombophilic

disposition. The angiotensinogen (A GT) gene has been studied with respect to

55

hemodynamic dysregulation. Polymorphisms in these three genes that have functional
effects on the gene in which they reside have all previously been investigated in relation
to PA (see review and meta-analysis(83)). Evidence of an association between FVL and
PA has been fairly consistent, while evidence of associations between MT HFR or AGT
variants and PA has been less convincing. Few studies have reported on these variants in
relation to PTD, and results to date have not demonstrated significant associations(84, 95,
97, 98, 100, 140). Other studies have attempted to link thrombophilias to vascular
placental pathology findings among placentas from complicated deliveries,(58, 101, 102)
but these have not compared findings with term or uncomplicated deliveries. Given that «
some vascular function genotypes have been linked to PA, and PA is certainly linked to
PTD, yet vascular function genotypes have not been convincingly linked to PTD or
placental histopathology findings, we hypothesized that relevant genotypes might be
associated with PTD subtypes defined by PA or hemorrhage-related placental pathology
findings. We aimed to assess the associations between maternal gene polymorphisms and

PTDs with clinical or pathology-based evidence of placental hemorrhage.

3.2. Methods
Study Protocol

The Pregnancy Outcomes and Community Health (POUCH) Study enrolled
women with singleton pregnancies (15-27 weeks’ gestation) from 52 clinics in five
Michigan communities. The POUCH Study protocol was approved by the Institutional
Review Boards of Michigan State University and Michigan Department of Community

Health, and all women provided informed consent. Women were eligible for the POUCH

56

Study if they had maternal serum alpha-fetoprotein (MSAF P) screening at 15-22 weeks,
no pre-existing diabetes outside of pregnancy, age 2 15 years, English language
proficiency, and no known congenital anomalies at the time of enrollment. Women with
unexplained high MSAF P (22 multiples of the median) were oversampled, constituting
7% of the cohort (estimated 3.5% in general obstetric population). At enrollment, women
provided a blood sample, participated in an in-person interview with a trained research
nurse, and filled out a questionnaire. From the interview and questionnaire, information
was gathered on demographics, anthropometrics, substance use, prenatal vitamin use, and
reproductive history. Women were asked to select their primary race or ethnic heritage
from a list, e. g. “White or Caucasian” or “Black or African-American” (referred to as
“white” and “black.
Subcohort sample

A subcohort was selected for detailed study of biologic samples and medical
records. The subcohort sample included all PTDs, all women with unexplained high
MSAF P, and a stratified sample of term pregnancies with normal MSAF P; black women
with term deliveries and normal MSAF P were oversampled. For the subcohort, medical
records were abstracted in detail by study nurses with labor and delivery experience,
placentas were examined by the study pathologist (PK Senagore), and genetic assays
were conducted. The current analysis includes 996 white or black subcohort women with
complete genotype and placental pathology data and no placenta previa.
Pregnancy outcome measures

Gestational age was calculated based on last menstrual period; this estimate was

replaced with an estimate from an ultrasound scan performed at <25 weeks’ gestation if

57

the two estimates differed by >14 days. PA was defined by evidence from chart review
by consensus of three clinician reviewers with labor and delivery experience. The case
definition required either (1) documented signs and symptoms consistent with PA (e. g.
significant bleeding not attributable to dilatation, uterine pain or tenderness, fetal
distress); or (2) retroplacental hematoma visualized on a prenatal ultrasound scan.
Placenta protocol

The pathologist was blinded to all clinical data including gestational age. For the
gross examination, parallel slices were made through the placental disc 1 cm apart. The
pathologist noted blood clots on the retromembranous, retroplacental, and disc cut
surfaces, and adjacent tissue changes, i.e. dissecting hemorrhage, infarcted or compressed
tissue, and red/brown tissue discoloration. Disc-impacting blood clot was defined as
gross examination evidence of a retro- or intraplacental clot impacting adjacent tissue.

For the microscopic examination, nine placental tissue samples were examined:
two membrane rolls, two sections of umbilical cord, and five full-thickness sections from
the disc(19), without pathologist knowledge of gross examination findings. Severe
histologic chorioamnionitis (HCA) was determined as previously described(19).
Microscopic vascular-related findings that fell within five constructs adapted from a
diagnostic coding tool were recorded(23). For our analysis, the construct of interest was
“Maternal Vascular — Disturbance of Integrity” (MV-I), which included microscopic
evidence of retroplacental blood with adjacent disc disruption/compression, decidual
hemorrhage in the basal plate, and decidual hemosiderin-like pigment in the membranes
or basal plate. High MV-I scores (i.e. the top quintile based on the distribution among

term, normal MSAF P deliveries) were previously shown to correlate with PTD risk. The

58

MV—I construct was selected to serve not as a ‘diagnostic instrument;’ but rather, as a
distributionally-defined measure that identifies a continuum of findings consistent with
maternal vessel bleeding. In this study, we considered two thresholds of MV-I scores to
serve as possible indicators atypical maternal vessel hemorrhage: the top decile and the
top quintile.

Thus, pathology-based evidence of placental hemorrhage includes gross
examination findings of a disc-impacting blood clot and/or high scores in the microscopic
examination MV-I construct. When PA cases are excluded, these findings are referred to
as subclinical evidence of placental hemorrhage.

Genetic assays

DNA was prepared from peripheral blood using a Puregene (Gentra) kit. Four
polymorphisms were assayed by Polymerase Chain Reaction (PCR) followed by
restriction digestion with appropriate restriction enzymes. The G-6A promoter variation
in AGT and the FVL variation in F5 were detected using published protocols.(141, 142)
The C677T polymorphism in MT HFR employed primers C677T (sense) 5’-TGA AGG
AGA AGG TGT CTG CGG GA-3 ’, (antisense) 5’-GAC GAT GGG GCA AGT GAT
TC-3’ for PCR amplification followed by digestion with Hinfl to produce 100 base pair
(bp) and 19 bp fragments for the C allele and 78, 22 and 19 bp fragments for the T allele.
The MTHFR A1298C variant assay employed primers A1298C (sense) 5’-TCT ACC
TGA AGA GCA AGT CC-3’, (antisense) 5’-CAC 'ITC CAG CAT CAC TCA CT-3’,
followed by MboII digestion of the PCR product to yield 72, 30, 28 and 20 hp products
for the A allele and 100, 30, and 20 bp products for the C allele. Minor allele frequencies

and deviation from Hardy-Weinberg equilibrium were calculated by race.

59

Analytic strategy

The current analysis includes white and black women with complete data on
polymorphisms and placental pathology, and no placenta previa. Analyses incorporate
weights (inverse of sampling probability) using the SURVEY procedures in SAS
(Statistical Analysis Software, Cary, NC) to account for the complex sampling scheme.

As a first step, we compared distributions of maternal characteristics to evidence
of placental hemorrhage using a 3-level hierarchical variable: (1) PA, (2) subclinical
evidence of placental hemorrhage (i.e. disc-impacting blood clots or the top decile of
MV-I scores) or (3) none. Second, with PTD as the outcome, we estimated race-specific
ORs for each variant assuming dominant models, i.e. women having 1 or 2 copies of the
minor allele versus with women having 0 copies, except when zero cells prohibited
analysis. This was repeated with PA as the outcome.

Next, we were interested in whether maternal genotypes were specifically
associated with the hypothesized placental hemorrhage-related PTD pathway. To this
end, race-specific polytomous logistic regression models were developed to compare two
broad PTD subtypes with term deliveries: (1) PTDs with any evidence of placental
hemorrhage (i.e. PA or subclinical evidence of placental hemorrhage), and (2) PTDs
without any evidence of placental hemorrhage. This analysis was repeated after changing
the threshold for defining a high MV-I score from the top decile to the top quintile.
Although few cases of preterm PA were available, we performed another polytomous
regression analysis afier separating PTDs with PA from PTDs with subclinical evidence

of placental hemorrhage. MTHFR analyses were repeated after excluding women who

60

were taking prenatal vitamins preconceptionally. All analyses were repeated after
excluding women with severe HCA.

Based on a type I error rate of 0.05, the POUCH Study was originally designed to
have at least 65% power to detect ORs of 2.0 in the subcohort for exposures present in
20% of the population, when considering PTD subtypes that occurred in at least 5-7% of
the population. The current analysis focuses on a narrower PTD subtype, which occurred
in only 2% of the population. Variation in allele frequencies and the necessity to stratify
on race resulted in 80% power to detect ORs ranging from 3.0 to 6.3 depending on
genotype and race (with the exception of FVL and AGT in blacks, where extreme

genotype distributions precluded most analyses.)

3.3. Results

Race-specific minor allele frequencies are shown in Table 3.1. The minor alleles
for MTHFR677, MTHFR1298, and FVL were more common in whites than blacks. For
AG T, the G allele predominated in whites while the A allele predominated in blacks.
Hardy-Weinberg equilibrium was not violated for any allele in either race.

The distributions of maternal characteristics in the study sample and the
prevalence of PA, subclinical evidence of placental hemorrhage, and no evidence of
placental hemorrhage by these characteristics are detailed in Table 3.2. The weighted
incidence of PTD was 10.7%. PA was clinically diagnosed in 30 women (2.1%), and
subclinical evidence of placental hemorrhage was present in 9.5% (as defined based in
part on a distributional cutpoint), and these were both overrepresented among PTDs

(7.3% and 12.3%, respectively). Other maternal characteristics did not differ significantly

61

across the outcome categories. Potential confounders of the genotype-pregnancy outcome
relations would need to be associated with both the genotypes and the outcomes. None of
the maternal characteristics met these criteria, thus these were not used to adjust
subsequent analyses.

Table 3.3 shows dominant genotype models for PTD. There were no significant
associations between any genotype and PTD. There was an association between FVL
GA/AA and PA (OR=5.8, 95% CI 1.1, 30.3) among white women (not in table).

Results of the polytomous logistic regression analysis of PTD subtypes defined
broadly as those with or without any evidence of placental hemorrhage (i.e. PA and/or
pathology-based evidence of placental hemorrhage) are presented in Table 3.4. Among
white women, those with PTD and evidence of placental hemorrhage were more likely
than women with term deliveries to have the FVL GA/AA genotypes (OR=4.8, 95% CI
1.6, 14.2) or the AGT GA/AA genotypes (OR=3.8, 95% CI 1.3, 10.5); the corresponding
ORs for PTDs without evidence of placental hemorrhage were close null (1.2 and 0.9,
respectively).

After changing the threshold of the MV-I construct from the top decile to the top
quintile (which identified 1/3 of all PTDs as having evidence of hemorrhage), results
were attenuated but still significant for F VL (OR=3.2, 95% CI 1.3, 8.3) but weaker for
AGT (OR=1.6, 95% CI 0.8, 3.1). Results for both MTHFR variant genotypes were null in
both blacks and whites. Repeating the MTHFR analyses in women who were not taking
prenatal vitamins prior to conception produced similar null results.

To ensure that infection-related PTDs were not obscuring true associations

b etween genotypes and hemorrhage-related PTDs, the models were also run after

62

excluding women with severe HCA from the dataset, and results were similar (not
shown).

Finally, we subdivided PTDs with evidence of placental hemorrhage into those
with PA and those with subclinical evidence of placental hemorrhage (Table 3.5). FVL
GA/AA was associated with both (PTD with PA OR=5.4, 95% CI 1.0, 28.2; PTD with
subclinical evidence of hemorrhage OR=4.4, 95% CI 1.1, 16.9). The AGT GA/AA
genotype was not significantly associated with PTD with PA (OR=2.5, 95% CI 0.5,
13.1), but was associated with PTDs with subclinical evidence of hemorrhage (OR=5. 1 ,

95% CI 1.5, 17.2).

63

 

Table 3.1. Race-specific minor allele frequencies for measured gene

polymorphisms among 560 white and 399 black women

 

Polymorphism

 

MTHFR(677) C>T
MTHFR(1298) A>C
FVL(1691) G>A

AGT(-6) G>A

dbSNP [D

Minor allele frequency

 

 

r31801131

r81801131

rs6025

rsSOSl

 

White Black
0.344 0.102
0.317 0.19

0.028 0.004
0.422 0.834

 

64

 

Table 3.2. Maternal characteristics of subcohort sample, and prevalence of placental

abruption, subclinical evidence of placental hemorrhage, and no evidence of

placental hemorrhage

Preterm delivery <37 weeks
Yes
No

Race
White
Black

Medicaid-insured
Yes
No

Age in years
<20
20-34
35+

Smoked during pregnancy

Yes (includes quit prior to
enrollment)
No

Cocaine use
Ever
Never

Pre-pregnancy Body Mass Index
(kg/m2)

<18.5

18.5 - 24.9

25-29.9

Z30

 

 

Subcohort
Sample
N %1'
959 100.0
223 10.7
736 89.3
560 75.4
399 24.6
516 45.7
442 54.3
154 13.3
735 78.4
70 8.3
270 26.7
689 73.3
80 8.7
879 91.3
40 3.3
424 45.7
218 24.5
277 26.4

65

Evidence of Placental Hemorrhage

 

 

 

Placental
Abruption
N %:l:
30 2.1
18 7.3
12 1.4
15 1.7
15 3.1
18 2.1
12 2.0
6 2.2
22 2.1
2 1.4
5 0.9
25 2.5
3 1.4
27 2.1
4 6.4
16 2.2
5 1.8
5 1.5

Subclinical
Evidence"r

N %:t
93 9.5
28 12.3
65 9.2
60 9.9
33 8.2
45 7.7
48 11.1
20 11.5
66 9.0
7 11.3
24 8.0
69 10.0
6 7.2
87 9.7
4 10.0
46 10.5
18 7.6
25 9.4

None

N %1
836 88.4
177 80.4
659 89.4
485 88.3
351 88.7
453 90.2
382 86.9
128 86.3
647 88.9

61 87.3
241 91.1
595 87.5

71 91.4
765 88.2

32 83.6
362 87.3
195 90.6
247 89.0

 

Table 3.2 (cont’d)

 

Pregnancy history
Nulliparous

Parous, no prior Preterm
delivery

Parous, prior Preterm
delivery

Hypertension
Yes (PE, PIH, or chronic)
None

Prenatal vitamin use
Started prior to pregnancy

Started during first trimester

Started prior to study
enrollment during
second trimester

Not taking at enrollment
(15-27 weeks)

Evidence of Placental Hemorrhage

 

 

 

Subcohort Pl t I S b Ii , 1
Sam 1e acen a u c mca
p Abrmion Evidence* None
N °/o1' N %x °/o;t N °/o:t
390 40.7 7 0.8 42 9.1 341 90.1
437 49.5 15 2.8 42 10.2 380 87.0
131 9.8 8 3.7 9 7.5 114 88.8
97 8.7 4 4.2 15 14.3 81 81.5
862 91.3 26 1.9 78 9.0 758 89.1
124 14.4 3 0.9 16 14.0 101 85.1
587 63.1 22 2.7 55 8.7 510 88.6
128 11.0 3 1.3 11 9.0 114 89.7
124 11.6 2 0.9 11 8.8 111 90.3

 

*Subclinical evidence of placental hemorrhage defined as presence of a disc-impacting blood clot in the

gross placental examination or a microscopic vascular - disturbance of integrity score in the top decile as

observed in the microscopic placental exam.

TColumn percent, weighted using inverse of sampling probability to reflect distribution in POUCH cohort.

IRow percent, weighted using inverse of sampling probability to reflect distribution in POUCH cohort.

66

 

Table 3.3. Association between vascular function genotypes (dominant
models) and preterm delivery among 560 white women and 399 black women

 

 

 

 

 

Preterm Delivery
Race Genotype
Yes No OR* 95% CI
White Total 1 52 408
MTHFR (677)
CC 62 174 1.0
TC or IT 90 234 1.0 0.7, 1.5
MTHFR (1298)
AA 70 189 1.0
AC or CC 82 219 1.0 0.7, 1.5,
FVL (1691)
GO 140 386 1.0
GA or AA 12 22 1.7 0.8, 3.7
AGT(-6)
GG 50 136 1.0
GA or AA 102 272 1.0 0.7, 1.6
Black Total 71 328
MTHFR (677)
CC 57 265 1.0
TC or T1" 14 63 1.1 0.5, 2.0
MTHFR (1298)
AA 48 220 1.0
AC or CC 23 108 1.0 0.5, 1.7
FVL (1691)
GG 71 325
GA or AA 0 3
AGT(-6)
GG 2 8 1.0
GA or AA 69 320 0.8 0.2, 3.8

 

*Odds ratios (OR) and 95% confidence intervals (Cl) from weighted (inverse of sampling
probability) logistic regression models.

67

 

Table 3.4. Association between vascular function genotypes (dominant models) and
PT D subtypes defined by presence or absence of evidence of hemorrhage compared
with term deliveries among 560 white women and 399 black women

 

 

 

 

 

 

 

 

Preterm Delivery Subtypes
Term With Evidence of Placental Without Evidence of
Hemorrhage* Placental Hemorrhage1Ir
Race Genotype n 11 OR* 95% CI n OR* 95% CI
White Total 408 28 124
MTHFR (677)
CC 324 13 1.0 49 1.0
TC or TT 236 15 0.8 0.4, 1.7 75 1.1 0.7, 1.7
MTHFR (1298)
AA 259 10 1.0 60 1.0
AC or CC 301 18 1.7 0.7, 4.0 64 0.9 0.6, 1.4
FVL (1691)
GG 526 23 1.0 117 1.0
GA or AA 34 5 4.8 1.6, 14.2 7 1.2 0.5, 3.0
AGT(-6)
GG 186 5 1.0 45 1.0
GA or AA 374 23 3.8 1.3, 10.5 79 0.9 0.6, 1.53
Black Total 328 18 53
MTHFR (677)
CC 322 16 1.0 41 1.0
TC or T1“ 77 2 0.5 0.1, 2.52 12 1.3 0.6, 2.6
MTHFR (1298)
AA 220 14 1.0 34 1.0
AC or CC 108 4 0.6 0.2, 1.9 19 1.1 0.6, 2.1
FVL (1691)
GO 396 18 53
GA or AA 3 0 0
AGT(-6)
GG 10 O 2
GA or AA 389 18 51

 

*Evidence of placental hemorrhage from clinical data or placental pathology exams. Preterm deliveries
With evidence of placental hemorrhage defined as preterm deliveries accompanied by placental abruption,
disc-impacting blood clot observed in the gross pathology examination, or a score in the top decile of the
microscopic vascular - disturbance of integrity construct in the microscopic placental pathology
examination. Preterm deliveries with evidence of placental hemorrhage defined as all other preterm
deliveries.
TOdds ratios (OR) and 95% confidence intervals (CI) from weighted (inverse of sampling probability)
polytomous logistic regression models (term deliveries = referent).
NOTE: Boldface denotes P<0.05.

68

 

 

 

 

 

 

 

 

 

 

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3.4. Discussion

In this study, we investigated MTHFR(677), MTHFR(1298), FVL, and AGT(G-
6A) variant genotypes in relation to PTD subtypes defined by evidence of placental
hemorrhage. Although none of the genotypes were associated with PTD overall, when we
brought information on delivery timing and placental hemorrhage together, we found that
FVL GA/AA and AGT(-6) GA/AA genotypes were both associated with PTDs having
evidence of placental hemorrhage in white women. This association was not attributable
solely to clinically—detected PA cases. Notably, the observed effects were specific in that
the ORs for PTDs without any evidence of placental hemorrhage were very close to the
null value.

A recent meta-analysis found a significant association between F VL and PA,
based on 10 studies.(83) Our results corroborate a strong association between FVL and
placental abruption risk, and contribute the additional finding that F VL is associated with
a specific subset of PTDs that have more broadly-defined evidence of placental
hemorrhage, including subclinical hemorrhage identified through placental pathology
exams. Normal pregnancy alters hemostasis, shifting the balance to a relatively
hypercoagulable state.(77) Inherited thrombophilias may exacerbate this shift and
predispose women to develop clots at the maternal-fetal interface. Although exact
mechanisms are unknown, these clots may somehow facilitate rupture of decidual blood
vessels, resulting in decidual hemorrhage and possibly also premature placental
detachment. However, subclinical decidual hemorrhage might lead to PTD via other

mechanisms.(107, 143)

71

We found that AGT(-6) GA/AA was associated with PTD with evidence of
placental hemorrhage among white women. The AGT(-6) promoter polymorphism is in
strong linkage disequilibrium with the AGT M23 5T polyrnorphism.(144) One study
conducted in Mexico identified an association between AGT M23 ST and preterm
premature rupture of membranes(95), and only two studies have reported on AGT
polymorphisms and abruption risk, with conflicting results.(45, 94) This variant has also
been associated with preeclarnpsia(145) and hypertension outside of pregnancy.(82)
Because PA has been consistently linked to high blood pressure,(73-76) it is unknown
whether the association between AGT genotypes and PA observed in one large study(45)
was attributable to hypertension. We speculate that local effects of the renin-angiotensin
system in the decidual spiral arteries(l46) may have implications for hemorrhage-related
PTD risk, possibly in the absence of systemic hypertension.

At the POUCH study’s outset, MTHFR was a promising candidate gene for
vascular diseases, and its variants have received a great deal of research attention in the
obstetrics literature since that time. We identified no associations between MTHF R
genotypes and placental abruption or hemorrhage-related PTD in black or white women.
These null results add to accumulating evidence that these variants may not be strongly
related to pregnancy outcomes(37, 38, 43, 57, 59, 90, 97, 140), although a few studies
have identified positive associations with PA or other poor pregnancy outcomes(36, 98,
100). While there is strong biologic rationale for a role of MTHFR variants in poor
pregnancy outcomes through a thrombophilia pathway, variant genotypes may only result
in hyperhomocysteinemia with a thrombotic tendency among individuals with low folate

intake. POUCH Study enrollment began after mandatory grain fortification in the United

72

States, and most women reported taking prenatal vitamins, thus true folate deficiency was
probably rare. Direct measures of folate and homocysteine status were not available in
the POUCH Study; however, we did not find any meaningful differences in our results
after excluding women who took prenatal vitamins prior to conception. We cannot rule
out a stronger effect in populations with substantially lower folate intake.

The results of this study suggest additional avenues for research. Variants in other
genes implicated in thrombophilias, e. g. Factor 2 (prothrombin) and plasminogen
activator inhibitor-1, and other variants in renin-angiotensin system genes, might be
associated with PTD with placental hemorrhage. Furthermore, given that the placenta has
a fetal genotype, consideration of fetal DNA and maternal-fetal genotype interactions
may shed additional light on risk of both PA and PTD.

The most important strength of this study is the objective assessment of gross and
microscopic placental pathology in a large sample of preterm and term deliveries, which
enabled us to investigate genotypes in relation to etiologically relevant PTD subgroups.
At least three studies have attempted to link maternal or fetal thrombophilias to specific
placental lesions within pregnancies complicated by PA, preeclampsia, or fetal growth
restriction; however, none of these studies included a comparison group of placentas
from uncomplicated pregnancies.(S 8, 101, 102) Given the prospective design, the
POUCH Study is limited by a relatively small number of PTDs with PA or other
evidence of placental hemorrhage. Some identified statistically significant relations, such
as that between FVL or AGT genotypes and hemorrhage-related PTDs in white women,
may be accompanied by inflated ORs, as is often the case for newly discovered

associations(147). While we believe the results of this study are valid and biologically

73

plausible, caution is warranted in interpreting the magnitude of the observed associations
in light of the acknowledged power limitations. No adjustments were made for multiple
comparions and it is likely that some identified associations would lose statistical
significance if we did so; however, in our view this practice is unwarranted in the context
of a priori selection of a modest number of candidate genes because it inappropriately
increases the probability of type II error(148).

Polymorphisms related to hemostasis and hemodynarnics may be associated with
PTD through pathways involving disrupted vascular integrity, ie, PA or subclinical
pathology-based evidence of placental hemorrhage. Pending replication of these findings
in other studies, it may be possible to identify a set of upstream markers including these
maternal gene polymorphisms that discriminates women at highest risk of vascular-
mediated PTD or overt PA, and to implement preventive measures to improve outcomes

for both the mother and child.

74

CHAPTER 4.

CLINICAL CHORIOAMNIONITIS, HISTOLOGIC CHORIOAMNIONITIS AND
EVIDENCE OF PLACENTAL HEMORRHAGE

4.1. Introduction

Infection and bleeding have been identified as two pathways to preterm delivery
(PTD)(3, 7), although these pathways may not be independent. Chorioamnionitis, or
inflamed fetal membranes, typically results fiom infected amniotic fluid and may be on a
causal pathway to PTD(149). Sometimes chorioamnionitis produces clinical symptoms
(i.e. clinical chorioamnionitis, CCA), but chorioamnionitis is more often subclinical and
can only be identified by histologic examination of delivered placental tissue (i.e.
histologic chorioamnionitis, HCA).

Early pregnancy vaginal bleeding and placental abruption (a severe bleeding-
related pregnancy complication) occur more frequently in pregnancies that culminate in
PTD (20, 21, 133, 138, 150-152). Several studies have linked placental abruption to
CCA(29), other clinical evidence of infection(24, 105, 111), or HCA(31, 40, 44, 112,
115), but other studies have not concurred(113, 114). Few studies have specifically
linked early bleeding with subsequent diagnosis of HCA or CCA(108, 109). Little is
known about the mechanisms that connect early bleeding to PTD, but some have
suggested that early bleeding is infection-related(108-110, 117).

Some evidence suggests that HCA and CCA are not necessarily concordant(153,
154), and we have identified no studies that consider both HCA and CCA in relation to
placental abruption in the same sample of pregnant women. Thus, it is unclear whether

associations with bleeding-related variables are similar for HCA and CCA.

75

Elevated maternal serum alpha-fetoprotein in the absence of fetal anomalies
(“unexplained” high MSAF P), measured in mid-pregnancy, is one of the most consistent
biomarkers of elevated PTD risk(6, 70, 71, 155). Mid—pregnancy high MSAFP levels are
also associated with early pregnancy bleeding(156) and placental abruption(3 1 , 52, 70,
71), but to our knowledge no studies have demonstrated an association with HCA. A key
goal of the prospective Pregnancy Outcomes and Community Health (POUCH) study has
been to understand what PTD pathways are marked by high MSAFP(6). The POUCH
study collected multiple types of evidence of placental hemorrhage ascertained through
mid-pregnancy maternal interviews, medical chart review, and placental histopathology
exams. The aims of this study were to compare HCA and CCA, and to evaluate risk of
HCA and CCA in relation to high MSAF P screening and evidence of placental

hemorrhage ascertained early and late in pregnancy.

4.2. Methods
Study Protocol

The Pregnancy Outcomes and Community Health (POUCH) Study enrolled 3019
pregnant women at 15 through 27 weeks’ gestation from 52 clinics in five Michigan
communities in 1998-2004. Women carrying singleton pregnancies with no identified
congenital anomalies at the time of enrollment were eligible if they were at least 15 years
old, had maternal serum alpha-fetoprotein (MSAF P) screening at 15 through 22 weeks,
did not have diabetes outside of pregnancy, and were proficient in English. The cohort
oversampled women with unexplained high MSAF P, such that these constitute 7% of the

cohort (estimated population prevalence, 3.5%). The study protocol received ethics

76

approval from Michigan State University and the Michigan Department of Community
Health, and all women provided written informed consent. At enrollment, women were
interviewed by a trained study nurse and filled out a questionnaire. These instruments
collected data on demographics, height and pre-pregnancy weight, reproductive history,
vaginal bleeding during the current pregnancy, medication use and substance abuse.
Women were asked to select their primary race or ethnic heritage from a list.
Subcohort Sample

A subcohort (N=1371) was selected for in-depth study, using a sampling scheme
designed to maximize efficient use of resources. The subcohort included all PTDs, all
women with unexplained high MSAF P (22 MoM), and a stratified sample of women with
term deliveries and normal MSAF P, with oversampling of Afiican-American women in
this latter category. Weighted analyses using the SURVEY procedures in SAS (version
9.1.3, Statistical Analysis Software, Cary, NC) were performed in order to appropriately
account for the sampling scheme, so that weighted frequencies can be interpreted in
relation to the cohort distributions (as prevalence or risk), additionally correcting for the
oversampling of high MSAF P into the cohort.
Chart Abstraction and Clinical Diagnoses

For the subcohort, prenatal and labor and delivery records were abstracted in
detail by study nurses. Gestational age was estimated by last menstrual period (LMP) and
by an ultrasound scan performed prior to 25 weeks’ gestation when available (97% of
subcohort). If the two estimates differed by more than 2 weeks, the ultrasound estimate
was used, otherwise the LMP estimate was used (79% of subcohort). PTD was defined as

delivery occurring prior to 37 completed weeks. Other information abstracted from

77

patient charts included episodes of vaginal bleeding and vaginal infections during
pregnancy, laboratory data including blood counts, and indicators of infection during
labor.

Placental abruption and CCA were determined based on signs and symptoms
recorded in patient charts. Placental abruption cases required either (1) significant
bleeding prior to delivery or intrapartum without other cause, abdominal or back pain,
uterine tenderness, or increased uterine tone; or (2) retroplacental hematoma visualized
on a prenatal ultrasound scan. CCA was defined as a documented fever >100F,
accompanied by at least two of the following four signs and symptoms: white blood
count >15,000, uterine tenderness, foul-smelling vaginal discharge or amniotic fluid, and
maternal or fetal tachycardia.

Gross Placental Pathology

For subcohort women, placentas were formalin fixed after delivery. Gross
examination was performed using standard methods. Parallel slices were made through
the placental disc 1 cm apart. Retroplacental or intraplacental blood clots visible on the
cut surface with evidence of adjacent tissue involvement, i.e. dissecting hemorrhage,
tissue infarction, compression, or red/brown discoloration were defined as disc-impacting
blood clots.

Microscopic Placental Pathology

A detailed microscopic placental examination was performed using nine tissue
samples per placenta: two membrane rolls, two sections of umbilical cord, and five full-
thickness sections from the disc(19), without pathologist knowledge of gross examination

findings. HCA was classified as severe (polymorphonuclear leukocyte inflammatory

78

pattern in chorionic plate and/or extraplacental membrane chorion and amnion, plus
karyorrhexis or necrotizing inflammation), mild (not meeting criteria for severe but
having at least one high-powered field with greater than 10 polyrnorphonuclear
leukocytes), or none. Microscopic vascular-related findings that fell within five
constructs adapted from a diagnostic coding tool were recorded. For the current analysis,
the construct of interest was “Maternal Vascular — Disturbance of Integrity” (MV-I),
which included microscopic evidence of retroplacental blood with adjacent disc
disruption/compression, decidual hemorrhage in the basal plate, and decidual
hemosiderin-like pigment in the membranes or basal plate. Findings were summed across
all relevant placental samples to derive a score for each woman. The MV-I construct was
selected to serve not as a ‘diagnostic instrument;’ but rather, as a distributionally—defined
measure that identifies a continuum of findings consistent with maternal vessel
hemorrhage. In this study, we considered the top quintile of MV—I scores to serve as a
possible indicator of atypical maternal vessel hemorrhage. An association between high
MV-I scores using this distributionally-defined cutpoint and PTD was previously
demonstrated(23).

Thus, two early and three late indicators of placental hemorrhage were
considered. The early indicators were (1) first trimester bleeding from maternal
interviews at 15-27 weeks (none, spotting only, or heavier bleeding) and (2) first and
second trimester bleeding documented in patient charts (none, first trimester only, second
trimester only, or both first and second trimesters). The late indicators were (1) placental
abruption; (2) disc-impacting blood clots identified in the gross pathology examination;

and (3) top quintile of MV-I scores from the microscopic pathology examination.

79

Analytic Strategy

The current study includes subcohort women with completed placental pathology
exams who identified themselves primarily as “White or Caucasian” (N=580) or “Black
or Afiican-American” (N =416). Univariate associations between categorical maternal
characteristics and HCA or CCA were determined using weighted frequency distributions
and Rao-Scott chi-square tests(132). Multivariable logistic regression models were
developed using forward and backward selection to identify maternal characteristics that
had significant independent associations with HCA or CCA.

We estimated the risk of HCA and CCA among women with high MSAFP and
early and late evidence of placental hemorrhage. For the CCA analyses, we simplified
information on bleeding in pregnancy to binary variables (any vs. none) in light of the
extremely limited number of cases meeting the criteria for CCA. We used weighted
logistic regression with HCA or CCA as the dependent variable to estimate odds ratios
(OR) and 95% confidence intervals (CI) for high MSAF P and early and late evidence of
placental hemorrhage. We adjusted for maternal characteristics that had been identified as
having significant independent associations with HCA or CCA in the previous step.

Although the sample size was limited, we explored two potential sources of
heterogeneity of effects for HCA: PTD and race. Interactions between high MSAF P,
evidence of placental hemorrhage and PTD or race were formally tested using likelihood

ratio tests (LRT).

80

4.3. Results
Descriptive Statistics

The distributions of PTD and maternal characteristics in the sample and the
prevalence of HCA and CCA according to these characteristics are shown in Table 4.1.
Overall, 238 (10.7%) women delivered preterm at <37 weeks, 77 (3.7%) delivered at <35
weeks, and 28 (1 .4%) delivered at <32 weeks. Approximately one-quarter of the cohort
was African-American, and 40.8% were nulliparous. Among subcohort women, 120
(weighted prevalence, 9.5%) had HCA and 17 (weighted prevalence, 1.4%) had CCA.
Both HCA and CCA were strongly associated with PTD at <37, <35, and <32 weeks.
Otherwise, HCA and CCA followed a different pattern of associations with maternal
characteristics. Higher risk of HCA was observed for women who were African-
American, had <12 years of education, were Medicaid-insured, were unmarried, or had a
vaginal infection during the index pregnancy in univariate analyses. Because many of
these characteristics are correlated, an adjusted model was developed (forward and
backward selection produced similar results). Two variables were significantly associated
with HCA in this model: race (African-American vs. white, OR=2.4, 95% CI=1.5, 4.0)
and education (<12 years vs. 212 years, OR=1.8, 95% CI=1.0, 3.2) (not in table). For
CCA, higher risk was observed for nulliparous women and smokers. When these two
variables were entered in the same logistic regression model, both remained associated
with CCA (nulliparous vs. parous, OR=8.6, 95% CI 2.6, 28.2; smoking, OR=4.2, 95%
CI=1.2, 14.5, ORs not in table).

Table 4.2 shows the associations between CCA and HCA overall and stratified by

PTD. Women with HCA had 2.6-fold greater odds of having CCA overall (95% CI 0.8,

81

7.9). This association was strong and statistically significant among PTDs (OR=7.2, 95%
CI 1.9 to 27.1). HCA and CCA were not associated among term deliveries (OR=0.8, 95%
CI=0.1, 6.7).
High MSAFP, Evidence of Placental Hemorrhage, and HCA

Table 4.3 shows the risk of HCA by high MSAF P and evidence of placental
hemorrhage, with unadjusted and adjusted ORs. Unexplained high MSAFP was
associated with HCA (aOR=1.7, 95% CI 1.0, 2.8). The absolute risk of HCA among
women with high MSAF P was 15.6%. First trimester bleeding ascertained during mid-
pregnancy maternal interviews was not associated with HCA. First trimester bleeding that
continued into the second trimester as recorded in patient charts was associated with
HCA (aOR=3.3, 95% CI 1.0, 11.6), although bleeding restricted to either the first or
second trimester was not. The absolute risk of HCA among women with bleeding in both
trimesters was 25.5%. None of the late evidence of placental hemorrhage was associated
with HCA. We included both MSAFP and first and second trimester bleeding in the same
model, and both remained significantly associated with HCA. Adjustment for race and
education changed the ORs for these variables by <10% (not shown).
High MSAFP, Evidence of Placental Hemorrhage, and C CA

Table 4.4 shows the risk of CCA by high MSAFP and evidence of placental
hemorrhage, with unadjusted and adjusted ORs. High MSAF P, early evidence of
placental hemorrhage, and high MV-I scores were not associated with CCA. Two late
indicators of placental hemorrhage —— placental abruption and disc-impacting blood clots —
were associated with increased risk of CCA. When we included placental abruption and

disc-impacting blood clots in the same model, both remained significantly associated

82

with CCA. Adjustment for smoking and nulliparity changed the OR for disc-impacting
blood clot by <10%, but it increased the OR for placental abruption by >10% (not
shown).

Subgroup analyses

We tested whether and the associations with HCA were similar for preterm and
term deliveries (not in table). The only significant interaction was between PTD and disc-
impacting blood clot (LRT p <.001). In term deliveries, there was non-significantly lower
risk of HCA for women with disc-impacting blood clots (3% vs. 8.9% in cohort term
deliveries, OR=0.3, 95% CI=0.1, 1.3). In PTDs, there was higher risk of HCA among
women with disc-impacting blood clots (29% vs. 14.5% in cohort PTDs, OR=2.8, 95%
CI=1.1, 7.4.

We tested whether the associations with HCA were similar for white and black
women (not in table). For high MSAF P and early evidence of placental hemorrhage,
there were no significant interactions with race. There were significant interactions
between race and late evidence of placental hemorrhage, i.e. placental abruption, disc-
impacting blood clot, and high MV-I scores (LRT p<.01, p<.001, and p=.01,
respectively). None of the 42 white women with HCA had placental abruption. Risk of
HCA among white women with disc-impacting blood clot (1.2%) or high MV-I scores
(3.2%) were somewhat lower than the background HCA risk in white women (6.9%).
Risk of HCA among black women with placental abruption (26.9%) or disc-impacting
blood clot (28.4%) were somewhat higher than the background HCA risk in black women
(17.4%), but HCA risk among black women with high MV-I scores was not elevated

(18.2%). In blacks, there were 1.8-fold greater odds of HCA among women with

83

placental abruption (95% CI 0.5, 6.1) and 2.0-fold greater odds of HCA among women

with disc-impacting blood clot (95% CI 0.8, 4.8).

84

 

Table 4.1. Distribution of selected maternal characteristics in study sample

and prevalence of histologic chorioamnionitis (HCA) and clinical
chorioamnionitis (CCA) according to maternal characteristics.

 

Total

Term Delivery
Preterm Delivery
<37 weeks
<35 weeks
<32 weeks

Race
Afiican-American
White

Education
<12 years

212 years

Medicaid Insurance
Yes
No

Marital Status
Unmarried
Married

Parity
Nulliparous

Parous

Smoked during
pregnancy

Yes
No

 

 

 

 

 

 

Subcohort
Sample

n %*
996 100.0
876 89.3
238 10.7

77 3.7

28 1.4
416 24.6
580 75.4
203 16.3
791 83.7
536 45.6
459 54.4
546 44.0
449 52.6
407 40.8
589 59.2
282 26.8
714 73.2

HCA
n °/oT
120 9.5
85 8.9
35 14.5
18 23.8
12 42.2
78 17.4
42 6.9
36 17.4
84 8.0
78 11.9
42 7.5
87 13.5
33 6.4
51 11.1
69 8.4
37 10.0
83 9.3

CCA

n o/o'l'
17 1.4
7 1.1
10 4.1
6 7.1
6 19.6
6 1.3 ,
11 1.4
5 2.3
12 1.2
1.3

1.5

7 1.1
10 1.6
12 2.9
5 0.4
7 3.0
10 0.8

 

85

 

Table 4.1 (cont’d).

 

 

 

 

 

Subcohort HC A C C A
Sample
n %* n %1' n %'l'
Vaginal infection during
pregnancy§
Yes 312 24.6 53 13.8 r 8 2.0
No 684 75.4 67 8.1 9 1.2
Age
<20 160 13.3 22 12.8 2 1.7
20-35 765 78.6 89 9.0 12 1.1
235 71 8.1 9 8.7 3 3.7
Pre-pregnancy BMI
Underweight 44 3.6 ' 7 13.9 2 7-0
Normal Weight 438 45.4 57 10.2 9 1.6
Overweight 228 24.7 23 8.2 6 1-7
Obese 286 26.3 33 9.0 0 0

 

*Column percentages, weighted to reflect sampling scheme.
TRow percentages, representing incidence of HCA or CCA in cohort, weighted to reflect sampling
scheme.

IP-value<.05 from Rao-Scott chi-square test.
§Includes Trichomonas vaginalis, Neisseria Gonorrhea, Chlamydia trachomatis, or bacterial
vaginosis

86

 

Table 4.2. Association between clinical chorioamnionitis (CCA) and histologic
chorioamnionitis (HCA) overall and stratified by preterm delivery, N=996.

 

 

 

HCA
Yes No OR* 95% Cl
All Deliveries
CCA Yes 6 11 2.6 0.8, 7.9
No 114 865
Preterm Deliveries
(<i7 Weeks)
CC A Yes 5 5 7.2 1.7, 27.1
No 30 198
Term Deliveries
e37 Weeks)
CCA Yes 1 6 0.8 0.1, 6.7
No 84 667

 

*Analyses weighted to reflect sampling scheme.
NOTE: Boldface denotes P<0.05.

87

 

Table 4.3. Association between histologic chorioamnionitis (HCA) and unexplained
_b_igh MSAF P and early and late evidence of placental hemorrhage.

 

 

 

 

n/row HC A Unadjusted Adj usted?
total risk °/°* OR* 95% CI OR* 95% CI
Total 120/996 9. 5
MSAFP
Unexplained high 23/159 15.6 1.8 1.1, 2.9 1.7 1.0, 2.8

EARLY EVIDENCE OF PLACENTAL HEMORRHAGE

First trimester bleeding from
midpregnancy maternal

interviews
None 94/776 9.9 1.0
Spotting only 16/113 11.3 1.2 0.6, 2.5
Heavier than spotting 10/ 107 8.4 0.9 0.4, 2.1

First and second trimester
bleeding from patient charts

None 83/729 8.9 1.0 1.0

First trimester only 21/159 11.5 1.3 0.7, 2.6 1.3 0.7, 2.5
Second trimester only 9/81 7.8 0.9 0.3, 2.2 0.9 0.3, 2.2
Both trimesters 7/27 25.5 3.5 1.1, 11.2 3.4 1.1, 10.9

LATE EVIDENCE OF PLACENTAL HEMORRHAGE
Placental Abruption 4/31 10.4 1.1 0.3, 3.5

Disc-impacting blood clot 9/62 8.3 0.9 0.4, 1.9

Maternal Vascular -
Disturbance of lntergrity
score (top quintile) 22/215 6.6 0.6 0.4, 1.1

'All analyses weighted to reflect sampling scheme.
+Model includes both indicators of placental hemorrhage that were significant 1n unadjusted model.
NOTE. Boldface denotes P<0. 05.

88

 

Table 4.4. Association between clinical chorioamnionitis (CCA) and unexplained
high MSAF P and early and late evidence of placental hemorrhage.

 

 

 

n/row CC: Unadjusted Adjusted?
l'lS
total W. OR* 95% CI OR* 95% CI
Total 17/996 1.4
MSAFP
Unexplained high 3/159 2.0 1.5 0.4, 5.6

EARLY EVIDENCE OF PLACENTAL HEMORRHAGE

First trimester bleeding
from midpregnancy
maternal interviews 5020 1.3 0.9 0.3, 3.0

First and second trimester
bleeding from patient
charts 6067 1.9 1.6 0.4, 5.7

LATE EVIDENCE OF PLACENTAL HEMORRHAGE
Placental Abruption 4/31 7.2 6.0 1.6, 22.8 4.4 1.3, 14.9

Disc-impacting blood clot 9/62 4.9 4.3 1.4, 13.4 3.6 1.2, 10.5

Maternal Vascular -
Disturbance of Intergrity
score (toLquintile) 22/215 0.7 0.5 0.1, 1.6

‘All analyses weighted to reflect sampling scheme.
*Model includes both indicators of placental hemorrhage that were significant in unadjusted model.
NOTE: Boldface denotes P<0.05.

89

4.4. Discussion

In this study, we found that HCA and CCA were not strongly concordant,
followed different patterns of maternal characteristics, and were associated with different
manifestations of placental hemorrhage. Two PTD risk markers observed in mid-
pregnancy — unexplained high MSAF P and bleeding in both first and second trimesters -—
were associated with findings of severe HCA in the delivered placenta, but were not
associated with CCA. CCA was associated with placental abruption and disc-impacting
blood clots in the delivered placenta, but these late indicators of placental hemorrhage
were not associated with HCA except in some subgroups of women, i.e. Afiican-
Americans and women who had preterm deliveries.

Many studies have reported that women with vaginal bleeding in early to mid—
pregnancy have increased risk of PTD, through mechanisms not yet fully explained(20,
21, 62, 67, 133, 136, 150-152). MSAFP, an early biomarker associated with risk of
PTD(70, 155), is also associated with early pregnancy bleeding(156). Our finding of
increased HCA risk among women with bleeding in the first and second trimesters or
high MSAFP suggests that these mid-pregnancy variables could actually mark an
inflammation-related pathway. Few prior studies have linked bleeding and high MSAFP
to measures of infection. A 2.6-fold increased risk of HCA but no increased risk of CCA
were reported among 33 women with first or second trimester bleeding compared with 63
non-bleeding controls in a research 1etter.(109) A larger prospective cohort study reported
that first tn'mester bleeding was associated with several vaginal infections and subsequent
development of CCA(l 08), a finding we did not replicate. We only observed an

association between bleeding and HCA when bleeding occurred in both the first and

90

second trimesters. A similar pattern has also been observed for vaginal bleeding in
relation to PTD in some studies.(20, 21) We suspect that persistent bleeding and high
MSAF P are both markers of ongoing placental problems that result from or support
infection.

Our results showed no association between HCA and any late evidence of
placental hemorrhage when all women were considered together. However, there was an
association between HCA and disc—impacting blood clots among PTDs, and there were
trends toward associations between HCA and both placental abruption and disc-
impacting blood clots among black women. Histologic evidence of inflammation is
objective and localizes inflammation to gestational tissues, but studies employing
placental examinations have not generated consistent associations with placental
abruption. Two cohort studies(40, 112) and two case-control studies(44, 115) identified
positive associations, two case-control studies found no association(113, 114), and one
cohort study had mixed (and overall very modest) results depending on the location of the
inflammatory infiltrate(3 1). We found that associations between HCA and late indicators
of placental hemorrhage differed by race, such that there was a trend toward increased
risk of HCA with placental abruption and disc-impacting blood clots in black women but
not white women. We find it plausible that populations with different underlying rates of
HCA could have different relations between HCA and placental abruption. We also
found that the association between HCA and disc-impacting blood clots differed by
timing of delivery, such that an association between these two variables was only
observed among PTDs. Differences in the results of studies to date may be attributable to

location or severity of inflammation, the use of blinded research protocols or clinical

91

pathology exams for assigning HCA status, selection of controls for case-control studies
(e. g. indicated preterm births, matching on parity), and study populations (e. g. hi gh-risk
or predominantly black population vs. general obstetric population). In addition,
variability in placental abruption case definitions (6. g. studying only preterm placental
abruption cases or requirement of a retroplacental clot) or differing criteria used to assign
HCA status might also help to explain different results between studies.

Although only 17 women in the study sample met the criteria for CCA, we
identified strong associations between this clinical measure of infection and both
placental abruption and disc-impacting blood clot, a gross lesion consistent with
premature placental detachment. We noted that HCA and CCA were strongly associated
with one another among PTDs, but not associated at term. Previous case-control studies
have reported imperfect correlation between these two manifestations of intrauterine
infection(153, 154). Our results are concordant with several prior studies that have linked
placental abruption with clinical evidence of infection, with measures of association
ranging from 1.55 to 9.71(24, 29, 105, 111). We speculate that a spurious association
between CCA and placental abruption could arise from the shared symptom of uterine
tenderness. Other signs and symptoms of CCA are not specific to infections in the
chorion and amnion (e. g. fever, leukocytosis) — these could indicate infection in other
gestational or non-gestational tissues, or even a maternal response to placental abruption.

Many studies have considered infection a risk factor for placental abruption(24,
40, 44, 105, 112, 115), but time order is not clear. A few studies have suggested that
bleeding during pregnancy might precede infection and (1) disrupt host defense

mechanisms to enable microbes to ascend from the lower genital tract, (2) provide a

92

nutritive substrate for bacterial growth in previously colonized gestational tissues, or (3)
allow microbes in the maternal circulation to gain access to gestational tissues and
spaces(108, 110). In the POUCH study, although bleeding and high MSAFP were
ascertained prior to determination of HCA status, it is not clear that disrupted
uteroplacental vascular integrity preceeded infection. High MSAF P, for example, could
be a marker of infection if infected membranes diffuse AF P more rapidly than healthy
membranes.(6) High MSAF P levels could also result from increased transfer across the
placenta attributable to a “breakdown in the placental fetal-maternal interface.”(155)
Bacteria may colonize the decidua and cause blood vessels to become fiiable, resulting in
clinically evident bleeding or high MSAF P, thus these might be indicators of existing,
otherwise clinically silent intrauterine infection.(108, 110, 117).

Generally, HCA results from bacteria ascending into the amniotic fluid, resulting
in neutrophil migration toward the fetal membranes. The definition of severe HCA used
in the POUCH Study incorporated information on number of neutrophils per high power
field in the fetal membranes as well as grade of infection (i.e. presence of karyorrhexis or
necrotizing inflammation). This definition previously identified women at markedly
increased risk of PTD,(1 9) and it is quite severe compared to other studies investigating a
link between HCA and placental abruption. It is possible that the inflammatory pattern
most useful in discriminating women at elevated PTD risk differs fiom the inflammatory
pattern that increases risk of (or results from) decidual hemorrhage or premature placental
detachment. Presence of karyorrhexis and necrotizing inflammation may imply that the
infection has been in place for some time (at least 24 hours). If placental abruption

precedes HCA and the observed association in some studies is attributable to neutrophil

93

 

recruitment to the site of bleeding, inflammation may not have sufficient time to reach a
high grade prior to delivery. The HCA definition used in this study did not incorporate
information on decidual inflammation, as some studies have.(109, 112) As a next step, it
may be helpful to consider acute and chronic decidual inflammation in relation to
evidence of placental hemorrhage. It is possible that inflammation in the decidua might
provoke a maternal response resulting in clinically evident infection more readily than
inflammation confined to the fetal membranes. Studying inflammation in the decidua
might help to reconcile our disparate results for HCA and CCA in relation to placental
abruption, as well as to help understand reasons for the limited concordance between
HCA and CCA.

Some limitations need to be considered when interpreting our findings. Given the
cohort design and general obstetric population studied, we had a limited sample of
placental abruption and CCA cases. We had 80% power to detect an OR of 3.3 between
HCA and placental abruption. It is possible that, by chance, we obtained a sample in
which no association between placental abruption and HCA existed (OR=1.1) when a
true association exists. We calculated that in order to have 80% power to detect an OR of
2.5 (similar to another cohort study)(40), we would need to recruit an additional 918
women and observe 5-fold increased odds of HCA with placental abruption in this group.
This is an extreme, but possible, scenario. We relied on data recorded in patient charts for
several key variables, including second trimester bleeding, CCA, placental abruption, and
vaginal infections in pregnancy. Given variability in prenatal practices, information may
not have been documented equally for all women, and some degree of misclassification is

possible.

94

Important strengths of this study include the availability for all women of an
objective placental pathology examination, a screening MSAF P determination, and
prospectively collected data on demographics, first trimester bleeding, and risk behaviors.
The determination of HCA used in this study was based on a detailed examination by a
pathologist who was masked to all clinical data and gross pathology findings. The
pathologist also identified disc-impacting blood clots and microscopic vascular-related
findings using a detailed descriptive protocol without any information on pregnancy
outcomes. The ability to generalize our results is also a strength, helped by sampling that
included women fi'om several communities and practices, and with both high and low
risk pregnancies.

This study provides limited support for the hypothesis that placental hemorrhage
in pregnancy is related to infection or inflammation. Future studies should consider that
associations between CCA or HCA and other variables may differ, because these'are not
the same entity. Reconciling differences between these manifestations of intrauterine
infection and their disparate relations with bleeding-related variables may be helped by

measuring deciduitis and its relations with CCA and HCA.

95

 

CHAPTER 5.

SUMMARY

The three studies presented in this dissertation have explored various facets of the
epidemiology of placental hemorrhage. First, we determined that four manifestations of
placental hemorrhage marked elevated preterm delivery risk, although they differed in
their associations with some maternal characteristics and were not highly concordant with
one another. Next, we found that women who were heterozygous for the Factor V Leiden
variant or the AGT G-6A promoter polymorphism were at increased risk of a specific
subset of preterm deliveries with evidence of placental hemorrhage, whereas they were
not associated with preterm deliveries that had no evidence of placental hemorrhage.
Finally, we found that early and late evidence of placental hemorrhage distinguished
between clinical and histologic chorioamnionitis: bleeding in the first and second
trimesters was associated with histologic chorioamnionitis, while placental abruption and
disc-impacting blood clots were associated with clinical chorioamnionitis. We also
identified some subgroup variations: associations between late indicators of placental
hemorrhage and histologic chorioamnionitis differed for blacks and whites and differed
between term and preterm deliveries.

Unexplained high MSAF P was featured in chapters 2 and 4. In chapter 2, we
confirmed previous observations that high MSAFP is associated with first trimester
bleeding and placental abruption. However, high MSAFP was not significantly associated
with either of the placental pathology findings indicative of placental hemorrhage,

although there were trends toward positive associations with both. In chapter 4, we found

96

that high MSAF P is associated with subsequent finding of HCA in the delivered placenta,
even after accounting for early pregnancy bleeding. Few maternal systemic biomarkers
have been identified for subsequent HCA. In a previous publication fi'om the POUCH
Study, associations between several circulating cytokines measured in mid-pregnancy
maternal plasma and preterm delivery at <35 weeks with HCA, but not with HCA
overall(157). The magnitude of the MSAFP-HCA association identified in this study is
modest (OR=1.7), and the predictive ability is likely to be quite low. However, the
association suggests that intrauterine infection may explain a portion of the consistent
association between MSAF P and preterm delivery.

Placental abruption and disc-impacting blood clots are two sources of evidence of
premature placental detachment. Findings from these three studies suggest that
considerable heterogeneity may exist in the causes of this outcome. Some cases of
placental abruption and disc-impacting blood clots may develop through thrombophilia or
vascular function pathways, such as those marked by Factor V Leiden variant and the
AGT(-6) GA/AA genotypes. Although we identified a very strong association between
clinical chorioamnionitis and both placental abruption and disc-impacting blood clots, it
is possible that the signs and symptoms of clinical chorioamnionitis actually occur in
response to abruption and bleeding. When all women were considered together, there was
no association between either measure of premature placental detachment and histologic
chorioamnionitis, although our results suggested that an association may exist among
black women or among women delivering preterm. Time order is not clear, and given the
strong association between early bleeding (in the first and second trimesters) and

subsequent HCA, it is possible that neutrophil infiltration occurs in response to bleeding.

97

However, it is also possible that in some subgroups of women, subclinical infection may
precede and cause decidual bleeding which may lead to a retroplacental hematoma or
overt placental abruption. A few studies have calculated an etiologic fraction for infection
and abruption, and these have been quite low (Kramer: 2.6%(40), Ananth: 2.4% for
preterm abruptions, 0.7% for term abruptions(24)). Thus, if infection is one cause of
abruption, it may be a very uncommon cause.

Ultimately, the three studies presented in this dissertation all attempt to provide
insight into pathways to preterm delivery that involve bleeding. Because associations
with maternal characteristics, gene polymorphisms, and intrauterine infection differed
between the measures of placental hemorrhage in many cases, we conclude that
considerable heterogeneity exists even within the “bleeding pathway.” Preterm deliveries
may be marked by early or late bleeding for a number of reasons, and some of these

reasons may overlap.

98

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