09 - Chapter 2

Review of Literature
REVIEW OF LITERATURE
2.1 Maternal and fetal outcome
2.2 Risk factors
2.3 Etiopathogenesis of preeclampsia
2.3.1 Placental implantation with abnormal trophoblastic invasion of
uterine vessel
2.3.2 Maternal maladaptive immunological intolerance
2.3.3 Oxidative stress and endothelial dysfunction
2.3.4 Genetic predisposition and epigenetic influences
2.3.5 Stage wise model of preeclampsia
2.4 Prediction of high risk women

2.5 Role of placental biomarkers in preeclampsia
2.5.1 Role of PAPP-A in preeclampsia
2.5.2 Role of free β-hCG in preeclampsia
2.6 Role of cytokines in preeclampsia

2.6.1 Role of TNF-α in preeclampsia
2.6.2 Role of IFN-γ in preeclampsia
2.7 Endothelial dysfunction in preeclampsia

2.7.1 Role of endothelin and nitric oxide in preeclampsia
2.8 Role of matrix metalloproteinases-9 in preeclampsia
2.9 Genetic polymorphism of MMP-9
Preeclampsia is pregnancy associated disorder, which is initially identifiable by new-

onset hypertension (systolic blood pressure 140/diastolic blood pressure 90 mmHg)
(ACOG, 2013). The current diagnostic clinical criteria for Preeclampsia are new-onset
hypertension and proteinuria after 20 weeks gestation (Sibai, 2003b; Weinstein,
2005). Preeclampsia aggravates very quickly from mild clinical manifestation to life
threatening conditions. Preeclampsia is heterogeneous in nature and includes multiple
organs. Along with hypertension and proteinuria multiple disorders like renal
insufficiency, liver function impairment, thrombocytopenia, pulmonary edema and
visual impairment (ACOG, 2013). Preeclampsia is a leading cause of adverse
obstetric outcomes worldwide (Sibai et al., 2003a).
Preeclampsia is a disorder, which is known to affect multiple organs of maternal

systems including cardiac, hepatic, renal, and nervous system. Preeclampsia
complicates 3%–8% of pregnancies in Western world and causes a major maternal
and fetal morbidity and mortality worldwide (Carty et al., 2010; Duley, 2009).
Preeclampsia and Eclampsia are responsible for 10%–15% of maternal deaths
worldwide (Duley, 2009). The incidence of Preeclampsia is three times higher in
pregnant women with a maternal history of this disorder (Zhang et al., 1997). The
incidence of Preeclampsia is reported 3% -7% in healthy nulliparous and 1% -3% in
multiparous women (Zhang et al., 1997).
2.1. MATERNAL AND FETAL OUTCOME
At present, the definitive cure for Preeclampsia is delivery of the placenta (ACOG
2002). Hypertension during pregnancy progresses aggressively from mild
hypertension to fatal medical condition. Delivery is often essential to minimize
maternal and fetal morbidity and mortality. Beside the progression of disease the
primary goal of obstetricians is to deliver the baby who is mature enough to adapt the
extrauterine environment without medical support (ACOG, 2008).
Hence, in preeclamptic pregnancies, the obstetricians have to balance the needs of

fetal growth in uterus and risk of disease progression to eclampsia, fetal growth
restriction and demise (Sibai et al., 2003a; Haddad and Sibai, 2005). Presently it is
recommended to deliver the fetus at 37 weeks regardless of disease severity (Knuist et
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al., 1998). Preeclampsia is chief contributor to the premature deliveries (Duley, 2009).
The mortality and morbidity rate is higher in preterm infants as compared to the term
infants (Engle et al., 2007; Kramer et al., 2000; McIntire et al., 2008). The gestational
age of 34 weeks is considered surrogate for delivery therefore deliveries occurring
between 34-36 weeks were believed to have lower risk of meeting any severe
morbidity in near or later future (Van Der Ham et al., 2012). But literature indicates
that these deliveries are at higher risk of having respiratory distress syndrome,
transient tachypnea, persistent pulmonary hypertension and respiratory failure as
compared to term infants (Dudell and Jain, 2006; Jain, 2008; Roth- Kleiner et al.,
2003; Ventilini et al., 2008; Engle et al., 2007). Preterm infants have nine times
higher incidence of respiratory distress syndrome than term infants (28.9% versus
4.2%) (Engle et al., 2007). Preeclampsia signifies a potential risk factor for
intrauterine fetal death with stillbirth rate of 21 per 1000 (Simpson, 2002). In severe
Preeclampsia the risk of fetal -death prevail over the pregnancy prolongation benefits.
However, in mild Preeclampsia, the risk of fetal death is less than 50% i e stillbirth
rate of 9 per 1000) (Simpson, 2002). In several studies Preeclampsia is associated
with fetal growth retardation. Fetal growth is a marker of fetal wellbeing. Odeogard et
al (2000) reported that infant born to any severe preeclamptic woman has 12% lower
weight than normal. Severe Preeclampsia is associated with bronchopulmonary
dysplasia (BPD) (Bose et al., 2009). The children exposed to the preeclamptic
pregnancy were reported to have low MDI (Major depression inventory) score at the
age of 2 years as compared to the normal pregnancy born children (Cheng et al.,
2004). Several studies report that preeclamptic gestation is the future window for the
development of endocrine, metabolic, cardiovascular and diabetic disorders (Wu et
al., 2009; Sommons et al., 2009).
In India tertiary antenatal care facilities are provided to pregnant women to ensure
early detection of clinical signs of complications such as new hypertension,
proteinuria, visual disturbance, symptoms of headache, epigastric pain, vomiting and
reduced fetal movements (Milne et al., 2009). Women with increased risk are
considered for assessments of blood pressure, urine samples, renal function, liver
function, uric acid, and platelet counts. Unfortunately, these frequent assessments are
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not executed in all parts of the India. The progression from mild hypertension to
eclampsia is 10-30 times more common in low-income nations than in high-income
nations (Duley, 2009).
2.2. RISK FACTORS
Evaluation of risk factors for developing Preeclampsia is important for optimising the
early prediction. Several studies have reported different risk factors. Odds ratio (OR)
or relative risk published based on reviews by Dekker and Sibai (2001). Important
risk factors have been mentioned in Table 1.
Table 1: Maternal risk factors for the development of preeclampsia
Factors Risk (Odds Ratio)

Age >40 years 2: 1
BMI >35 kg/m2 2: 1

Nulliparity/ primiparity 3: 1
Preeclampsia in a previous pregnancy 7: 1
Family history of preeclampsia 3: 1
According to WHO (World’s Health Organization) 20% of preterm birth reported

each year are associated with Preeclampsia (Kinney et al., 2012; Liu et al., 2012).
This condition is more important in developing nations where the incidence of
hypertensive disorders during pregnancy is 20 times higher (Walker, 2000; Roberts
and Lain, 2002).
The classical risk factors of Preeclampsia are primi-pregnancy, maternal age, insulin
resistance, insufficient nutrient (calcium) intake, metabolic syndrome, immune factors
and genetic predispositions (Duckitt and Harrington, 2005; López-Jaramillo et al.,
2005). The risk factors of Preeclampsia are Population, race and Country dependent
(Xiao et al., 2014; López-Jaramillo et al., 2005). Unhealthy life style with low cost
high calories food causes metabolic dysregulation and maternal obesity (Monteiro et
al., 2004). Maternal health and nutrition are vital as it is a regulatory risk factor that
can be incorporated to avert adverse obstetric outcomes, predominantly in developing
countries (Abu-Saad and Fraser, 2010).Women with poor nutrition intake are
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predisposed to gestational complications like Preeclampsia and gestational diabetes

(Roberts et al., 2003). According to Barker (2002) hypothesis, utero fetal
programming due to poor maternal health and malnutrition during gestation may
affect the fetal organ development and fetal growth. Many epidemiological studies
established the fact the poor maternal health results in development of disorders like
pregnancy hypertension, metabolic syndrome, gestational diabetes and vascular
disease (López-Jaramillo et al., 2010; Moore et al., 2004; Brker et al., 2002).
Prevalent factors of Preeclampsia development are associated with obesity, insulin
resistance and 5in developed countries whereas ethnicity, maternal malnutrition and
socioeconomic status in developing countries (Lopez Jaramillo 2010; Lui et al.,
2012). Low maternal education and low-income population has reported to have
higher risk of Preeclampsia (Ota et al., 2014).
Maternal age is found to be an associated risk of Preeclampsia (Li et al., 2016; Villar
et al., 2006; Poon et al., 2010). Women with maternal age above 35 or 40 are at
higher risk of developing Preeclampsia (Dukkit and Harrington, 2005). Older age is
associated with glucose intolerance due to reduced insulin sensitivity (Fulop et al.,
2003). Therefore, insulin resistance could be a contributor to the higher risk for
Preeclampsia in older women. Obese women have higher risk of pregnancy
hypertension, which may also be related to insulin resistance. Studies have shown that
elevated levels of free fatty acids in obese people lead to insulin resistance and
compensate hyperinsulinemia by increasing blood pressure (Wang et al., 2013; Wolf
et al., 2004). Medical history of chronic hypertension (Li et al., 2016), high BMI
(Wang et al., 2013; Li et al., 2016) and severe anaemia increased the risk of
Preeclampsia by three times. Having cardiac or renal disorder, diabetes, nulliparity
and maternal age >30 years doubles the risk of developing Preeclampsia (Li et al.,
2016). Beside these markers women with antiphospholipid syndrome and women who
conceived by in-vitro fertilization has higher chances of developing the disease
(Wright et al., 2012). Amongst the protective factors, improved maternal diet, more
than 8 antenatal visits and reduction in maternal weight are significantly associated
(Li et al., 2016; Gudnadottir et al., 2016).
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The classification of the pregnancy hypertension disorder is provided by the NHBPEP

(National High Blood Pressure Educational Program) (Program NH, 2000). NHBPEP
has classified the disorders of the pregnancy hypertension in the following four
groups:
1. Gestational hypertension
2. Preeclampsia and Eclampsia Syndrome
3. Preeclampsia syndrome superimposed on the chronic hypertension
4. Chronic hypertension
Gestational hypertension → Formerly termed as the pregnancy induced

hypertension. If Preeclampsia syndrome does not develop and the hypertension
resolves after 12 weeks postpartum then it is called gestational hypertension. It is re-
designated as transient hypertension.
Preeclampsia → Preeclampsia is characterized by the hypertension and significant

amount of protein in urine, which occurs after the 20th week of gestation in women
who are not previously known to be hypertensive. Other signs and symptoms include
edema and headache and in severe cases, the condition is associated with seizures,
liver and kidney dysfunction as well as clotting abnormalities, adult respiratory
distress syndrome and fetal growth restriction (FGR).
Eclampsia The onset of the convulsion in women with preeclampsia that cannot be
attributed to other causes is called as eclampsia. The seizures are generalized and may
appear before, during or after the labour.
Chronic hypertension→ Hypertension diagnosed before 20th week of gestation and

persists even after 12 weeks postpartum.
Preeclampsia syndrome superimposed on the chronic hypertension→ New onset

of proteinuria after 20th week of gestation in a chronic hypertensive women
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Diagnosis of the pregnancy hypertensive disorders:
Gestational hypertension • New onset of hypertension (140/90mmHg)

• No proteinuria
• BP returns to normal after 12weeks postpartum
• Final diagnosis made only postpartum
Preeclampsia Mild Preeclampsia:

• BP ≥140/90 mmHg after 20th weeks of gestation.
• Proteinuria ≥300mg/24 h
Severe preeclampsia:
• BP ≥ 160/110 mmHg
• Proteinuria 2.0 gm/24h
• Platelets <100,000/µL
• Persistent headache and epigastric pain
• Visual disturbance
Eclampsia • Seizures in preeclamptic women that cannot be attributed to
other cause.
Superimposed • New-onset proteinuria ≥300mg/24h in hypertensive women
Preeclampsia but no proteinuria before 20th weeks´ gestation.
• A sudden increase in proteinuria or blood pressure or
Platelets <100,000/µL in women with hypertension and
proteinuria before 20th weeks´ gestation
Chronic hypertension • BP ≥140/90 mmHg before pregnancy or diagnosed before
20th weeks of gestation not attributed to the gestational
trophoblastic disease. OR
• Hypertension first diagnosed before 20th weeks gestation and
persistent after 12 weeks postpartum
The disease etiopathogenesis starts much earlier than the symptom appears. Currently
available criteria diagnose the disease after 20th weeks of gestation. Early intervention
can improve the maternal and fetal outcome. Early prediction of high risk women can
minimize the associated adverse outcomes.
2.3. ETIOPATHOGENESIS OF PREECLAMPSIA
Bell (2010) has reviewed the history of preeclampsia. Leon Chesley is considered the
father of Preeclampsia research. He is known for his extensive renal function studies
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and prospective follow up of eclamptic women for 43 years. He is credited as an

inspiration for genetic studies in reference of Preeclampsia. In 1739, De Sauvages
used the term, “eclampsia”, for the first time and differentiated between seizures of
eclampsia and epilepsy. He also described that eclampsia was acute in nature and
resolves after removal of placenta. Proteinuria in eclampsia, was introduced by John
Lever in his text book "Diseases of the Kidney" in 1843. By the middle of 18th
century headache, stomach pain, loss of vision and swelling in the upper body were
recognized as hallmark of Preeclampsia. However, the introduction of blood pressure
measurement for recognition of Preeclampsia was done by Scipione Riva-Rocci’s
mercury manometer. Since then new onset of hypertension and proteinuria have been
the major signs used in the classification of preeclampsia.
De Sauvages in 1739 gave his view on the causation of Preeclampsia. He

hypothesized that the convulsion related to Preeclampsia results from the mechanism
by the nature, which is activated to free the maternal body from any morbid element.
In 1821 Thomas Denman proposed the theory of cerebral congestion. He posited that
Preeclampsia risk is directly linked to uterus. Expanded uterus exerts greater pressure
on blood vessels during pregnancy. This increased pressure on blood vessels results in
overload of cerebral vessels and related convulsions. In 1849, Smith challenged the
theory of cerebral congestion and suggested that pregnancy is the physiological state
of increased blood circulation. Smith pointed that there would be more cases if uterus
was the cause of Preeclampsia convulsion. Smith’s theory revolves around the toxic
elements. He believed that healthy pregnancy is directly associated with elimination
of wastes from the maternal system. If maternal system fails to eliminate the waste it
will accumulate and results in toxaemia. Along with toxaemia many other causes
which included excessive stimulus to the spinal centres by the mechanical or
emotional stress, bloodletting, atmospheric changes like temperature and wind and
irritation of abdominal passages like uterus, uterine, intestine and stomach. Another
theory of Preeclampsia development was proposed by Roberts in 1989. Roberts’s
theory posited that Preeclampsia is an endothelial disorder. They described that poor
trophoblast invasion results in reduced placental perfusion. Placental hypoperfusion
makes placenta ischemic, which releases morbid factors in maternal circulation. These
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morbid factors were known to cause the endothelial damage and subsequent
symptoms.
Redmen and Sargent proposed two stage model for disease development in 2005
(Redmen and Sargent, 2005). Two stage model was accepted by majority of the
scientific committee (Hlaudinwich et al., 2007; Roberts and Gammill, 2005; Roberts
et al., 2009). Redmen and Sargent, (2005) said that poor trophoblast implantation
results in hypo-perfusion of placenta (Stage-I) which leads to maternal syndrome
(Stage-II) (Fig-1). Subsequent progress from stage I to stage II is still a quest for
intense research.
Two-stage model given by Redmen is well explanatory but some modifications were
introduced by Roberts and Hubel (2009) for better understanding of disease
development. Firstly, each stage has different set of biomarkers. Secondly,
biochemical substances produced by the hypo perfused placenta is not a ‘‘toxin’’ but
rather the result of an adaptive fetal response (Roberts and Hubel, 2009).
STAGE-I
Impaired implantation STAGE-II
Pathway?? Maternal Syndrome of
Leads to placental
Hypo-perfusion Hypertension
Figure 1: Two-stage model of Preeclampsia: Preeclampsia is initiated by Impaired

implantation provokes release of morbid factors (Stage-I). These release of morbid
factors results in maternal pathophysiological symptoms (Stage-II) (Modified:
Roberts and Hubel, 2009)
Previous studies suggest that abnormal placentation is taking place before the stage of
vessel remodelling (Huppertz, 2008). Roberts and Hubel (2009) pointed out, whether first
trimester markers are sensitive to Preeclampsia prediction specifically or is associated
with any other disorder. From the placental bed biopsies it is evident that placental protein
expression is found to be altered in IUGR pregnancies. Altered expression of placental
proteins is the indication of abnormal placentation (Burger et al., 2004; Plasencia et al.,
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2007; Rovere-Querini et al., 2006; Vatten et al., 2007; Cetin et al., 2006; Ong et al.,
2000; Spencer et al., 2007). Abnormal uterine artery Doppler in second trimester of
pregnancy indicates increased vascular resistance due to failed remodelling of the vessels
of the intervillous space (Chien et al., 2000). Studies of placental bed biopsies with
abnormal uterine artery Doppler revealed that vascular remodelling is not unique to
Preeclampsia (Aardema et al., 2001). These findings show that stage –I markers are not
associated with Preeclampsia only, but can lead to other disorders as well and stage-I is
not sufficient to cause Preeclampsia. The expression of biomarkers like antiangiogenic
factor s-Flt (Shibata et al., 2005), cellular fibronectin (Powers et al., 2008), or leptin
(Catov et al., 2007) is altered in Preeclampsia but not in IUGR. These findings suggest
that these pathways specifically involved in the development of Stage-II of Preeclampsia.
The linkage between placental (Stage-I) and maternal (Stage-II) are the factors that
modify maternal metabolism and acts upon placenta to compensate fetal nutrient
requirement and facilitate nutrient transfer. Women who fail to acquire such modification
would be more likely to develop Preeclampsia. This hypothesis was evident by the
finding that 70% of infants born to preeclamptic mothers are not growth restricted. It is
possible that non-growth restricted pregnancies do not have failure of vascular
remodelling (Li et al., 2016). From the data available, it is suggestive that impaired
placentation is associated with other complications like IUGR along with Preeclampsia.
The difference is originated after abnormal placentation, which is actually a linker
between placentation and maternal symptoms such as hypertension and proteinuria.
Till 20th century the etiopathogenesis was unsolved, but intense efforts of researchers
have made a clear vision of Preeclampsia development. Instead of one disease
preeclampsia appears to be a culmination of factors that likely involves a number of
maternal, placental, and fetal factors. Currently considered important factors are as
follows:
• Placental implantation with abnormal trophoblastic invasion of uterine vessel.

• Immunological maladaptive tolerance between maternal, placental, and fetal tissues.
• Oxidative stress and endothelial dysfunction
• Genetic factors including inherited predisposing gene as well as epigenetic
influences.
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2.3.1. Placental implantation with abnormal trophoblastic invasion of uterine vessel
The placenta is a crucial organ, which develops in uterus during pregnancy. The
function of placenta is to provide oxygen and nutrients to the growing fetus and
eliminating waste products from foetus’s blood. Placenta is attached to the uterus
wall, and umbilical cord arises from it. Human placenta is dark reddish-blue or
crimson in colour. It is 9 inches long and 0.8–1 inches thick. The thickness is highest
in the centre, which gradually decreases toward the edges. Placenta weighs
approximately 0.5 kg. The umbilical cord arising from placenta is approximately
22–24 inches in length and has two umbilical arteries and one umbilical vein. The
umbilical cord is inserted into the chorionic plate by a peculiar connection. Umbilical
vessels are subdivided over the placental surface and form a network, which is
covered by a layer of cells. The vessel network forms a villous tree structures.
The placental development is initialized on implantation of the blastocyst into

endometrium of uterus wall. The outer layer of the blastocyst forms trophoblast,
which is the outermost layer of the placenta. Trophoblast is further divided into two
layers: the inner layer cytotrophoblast and outer is syncytiotrophoblast.
Cytotrophoblast cells undergo the differentiation and fusion process to form
multinucleated cell layer of syncytiotrophoblast, which covers the surface of the
placenta. The syncytiotrophoblast contributes to the barrier function of the placenta.
Successful embryo implantation is consequent result of parallel and synchronised

development changes occurring in the embryo and endometrium during the
preimplantation phase (Banerjee and Fazleabas, 2009; Van et al., 2009).
Endometrium is innermost layer of uterus. The cascade of events of morphological,
biochemical and physiological changes, which are acquired by endometrium before
receiving the embryo, is called as decidualization. The process of decidualization is
initiated by the coordinated, progesterone dependent embryonic signalling in the
endometrium. The fundamental factors of embryonic signalling involve proteases,
growth factors, adhesion molecules, cytokines and transcription factors. All these
factors implicate the biochemical and morphological changes in the endometrium
(Modi and Godbole, 2009; Nimbkar- Joshi et al., 2009). A whole genome study was
conducted to decode the impact of embryo derived hCG factors on the endometrium
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tissues. It was found that hCG stimulates the expression of trophoblast invasion
promoting factors such as leukemia inhibitory factor (LIF) (Stewart et al., 1992; Tapia
et al., 2008), C3 the complement component, matrix metalloproteinases (MMPs),
superoxide dismutase-2 (SOD and glycodelin. Hormone hCG is also involved in the
suppression of embryonic developmental Wnt-signalling pathway and soluble frizzled
receptor protein 4. hCG is involved in suppression of oxidative stress by C3,
glycodelin and SOD2. MMPs aids endometrium ECM remodelling during the
implantation (Goffin et al., 2003). hCG influence the expression of MMP-7 which
would possibly promote the trophoblast invasion (Sherwin et al., 2007; Yanaihara et
al., 2004). Embryonic signalling promotes the morphological changes in the
endometrium. In response to embryo signalling functional endowment occurs in
endometrium which promotes the trophoblast invasion. Endometrium is sensitive to
quality of embryonic signalling. In presence of poor embryonic signalling the function
endowment of endometrium leads to trophoblast invasion inhibition.
Figure 2: Embryonic signal produced at feto-maternal interface stimulates morphological

changes and functional changes in endometrium: As the result of embryonic signals,
process of decidualization and angiogenesis is initiated. In parallel, the endometrium
advances its capability to differentiate a good vs. poor embryo. It also generates an
inflammatory environment, which may supports invasion and commands the decidual
immune cells to avoid fetus refusal. Source: Modi et al., 2012)
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A healthy pregnancy includes changes in cardiac output to meet the nutrient needs
and waste disposal of growing fetus (Redman and Sargent, 2009). These changes take
place in first trimester. During this phase, cytotrophoblast cell of trophoblast invade
the maternal decidua and then the myometrium. This invasion leads to vascular
modification of maternal spiral arteries into low resistant and high blood flow vessels.
These modified maternal spiral arteries are capable to meet the nutrient requirements
of growing fetus. If somehow the cytotrophoblast cells of trophoblast fail to invade
the decidua then there will be incomplete invasion in myometrium. Incomplete
invasion results in impaired modification of spiral arteries (Bell, 2010).
Figure 3: Abnormal Spiral artery remodelling and placentation in preeclamptic

Pregnancy: In normal pregnancy, placentation, cytotrophoblasts invades the
maternal spiral arteries and transforms them from small-capacity high resistance
vessels to high- capacitance low resistance vessels for providing adequate
placental perfusion to endure fetus growth. At the time of invasion, the
cytotrophoblasts is discerned from an epithelial phenotype to endothelial
phenotype. This discerning is known as a “pseudovasculogenesis” (upper
section). In preeclamptic pregnancies, the process of pseudovasculogenesis is
disrupted and cytotrophoblast cells fail to adapt invasive phenotype. Which
results in shallow spiral artery invasion and incomplete modification to high-
capacitance low resistance vessels (lower section). (Source: Robert et al., 2009)
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In turn there will be restricted blood flow through the placenta, which limits the
nutrient and oxygen supply and waste elimination through placenta. To compensate
the blood flow deficiency, maternal system raises the blood pressure, which is the first
indication of Preeclampsia. This hypertension is resolved after delivery, which
suggests Preeclampsia is placenta originated disease (Redman and Sargent, 2005).
This concept was supported by several studies in which placental physiology of
pregnancies affected by Preeclampsia was compared with the pregnancies unaffected
by Preeclampsia (Bell, 2010).
2.3.2. Maternal maladaptive immunological tolerance
During pregnancy, maternal organ system acquires several changes for a successful
and healthy pregnancy. Like cardiac output, the maternal immune system also needs
to undergo reversible alterations. During normal pregnancy, there is absence of
maternal immune response towards the growing fetus and placenta, which is called
immunological tolerance (Kanellopoulos-Langevin et al., 2003). Maternal
immunological tolerance is vital for establishment of pregnancy (Redman and
Sargent, 2009) and trophoblast invasion (Kanellopoulos-Langevin et al., 2003).
For a successful pregnancy, appropriate implantation and placentation are vital (Red-
horse et al., 2004; Sargent et al., 2006). Any variation in these two events may cause
immediate or long-term complications such as IUGR, IUD, spontaneous abortion,
infertility and preeclampsia. During pregnancy the proliferation and migration of
cytotrophoblast cells leads to successful vascular remodelling (Laresgoiti-Servitje et al.,
2010). After 8 weeks of gestation cytotrophoblast differentiates into syncytiotrophoblast
and extravillous trophoblast (Laresgoiti-Servitje et al., 2010). The extravillous trophoblast
is highly proliferative and invasive which invades placenta and helps in vascular
remodelling (Zhou et al., 1997; Laresgoiti-Servitje et al., 2010). In case of normal
pregnancy extravillous trophoblast expresses class I MHC molecules such as HLA-E, F
and G (Ishitani et al., 2003) which are not recognised by the T helper cells and fetus
remains protected from maternal immune system. HLA-G plays an important role in
maternal immune system regulation during pregnancy (Hviid, 2006). The genetics of
HLA-G has been found to be associated with etiopathogenesis of Preeclampsia (Hviid,
2006). It has been postulated that reduced expression of HLA-G in placenta is associated
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with development of Preeclampsia (Hylenius et al., 2004). Several research studies have
confirmed the downregulated expression of HLA-G mRNA in placenta of Preeclampsia
patients (Goldman-Whol et al., 2000). In contrast to these findings, Shobu et al (2006)
found no significant difference in HLA-E, F and G expression among Preeclampsia and
normotensive placentas.
Other immunological theory associated with Preeclampsia is Th1/Th2 shift (Hu et al.,
2007; Saito and Sakai, 2003). According to this theory, in normal pregnancy this
immunological balance shifts towards Th2 due to the presence of progesterone and
anti-inflammatory cytokines such as IL-10 and 1L- 4 (Piccini et al., 2000). However,
in preeclampsia patients the expression of IFN-γ, which is a pro-inflammatory
cytokine, is upregulated and IL-10 and 1L-4 is downregulated (Arriaga- Pizano et al.,
2005). Increased IFN- γ promotes Th1 response. Th1 cytokines inhibits Th2 response
(Laresgoiti-Servitje et al., 2010). Saito and Sakai (2003) has reported that Th1
subpopulation is common in Preeclampsia patients.
Figure 4: Trophoblast invasion regulation by paracrine factors: The signal exchange

between endometrium and blastocyst is pivotal for implantation. Endometrium cells
such as uNK, macrophages and stromal produces numerous growth factors and
cytokines to regulate trophoblast invasion. The pro-invasive paracrine factors
promotes invasion are: interleukins 1,6, 8 11, 15 and LIF; Growth factors EGF, HGF,
IGFBP-1 etc. The anti-invasive paracrine factors inhibits trophoblast invasion are:
TGF-β, TNF-α, IFN-γ, Kisspeptin etc. Source: Modi et al., 2012)
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Endometrium cells secrete several types of biochemical factors such as cytokines and
growth factors, in response to the embryonic signalling by blastocyst. These factors
may positively or negatively influences the trophoblast invasion by paracrine
regulation. The factors secreted by endometrium are sensed by trophoblast.
Trophoblast strategizes the programming of invasion and implantation on the basis of
factors produce by endometrium.
Specific mechanisms which promote development of Preeclampsia are yet to be

described, but among the proposed mechanisms, impaired trophoblast invasion is
mostly accepted which initiates the Preeclampsia etiopathogenesis by reducing the
spiral artery remodeling and vascularity of the placenta. The maternal immune system
appropriately regulates progression of trophoblast invasion and spiral artery
transformation (Lala and Chakraborty, 2003; Wells et al., 2007). The decidua which
is the lining of the uterus forms the maternal share of the placenta and have several
immune cells like Macrophages, natural killer (NK) cells, dendritic cells (DCs), T-
cells and T regulatory cells (Tregs), which are required for proper migration of
trophoblast cells through the uterus (King, 2000; Trundley and Moffett, 2004;
Williams et al., 2009). These cells penetrate the decidua and assemble around the
trophoblast cells letting them to reach the endometrium through controlled removal of
immune cells in the spiral artery (Herberts et al., 2010; Lash et al., 2010; Nagamatsu
and Schust, 2010; Silva et al., 2008). Uterine NK cells (uNK), macrophages and DCs
has an essential role in trophoblast invasion and decidual formation (Cohen et al.,
2009; Hanna et al., 2006). T-regulatory cells and regulatory cytokines confirm the
appropriate control and function of pro-inflammatory cells for accurate invasion
(Trundley and Moffett, 2004; Somerset et al., 2004; Tilburgs et al., 2008). Th2
dominating state is promoted by DCs to encourage immune tolerance of the maternal
immune system for the fetus (Miyazaki et al., 2003). The decidual immune cells
during pregnancy work together to safeguard proper implantation and trophoblast
invasion that is neither shallow nor overly invasive (Lala and Chakraborty, 2003;
Wells et al., 2007). For a healthy pregnancy, the balance of immune cells and the
factors they produce are vital, any imbalance could potentially result in impaired
placental vascularization remodeling, which increases the risk of Preeclampsia or
pregnancy loss (Redman and Sargent, 2010, Van Nieuwenhoven et al., 2003).
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In contrast to normal pregnancies, preeclamptic pregnancies are associated with

inappropriate immune responses, which include alternately activated immune cells
and cytokines (Freeman et al., 2004; LaMarca et al., 2013). Placental ischemia which
results from incomplete or impaired trophoblast invasion has been suggested to create
an imbalance in immune function that promotes chronic inflammation which is
similar to an autoimmune disease (Redman and Sargent, 2010; Cornelius et al., 2013;
Irani et al., 2010). This immune imbalance is between pro-inflammatory immune cells
and cytokines; and anti-inflammatory immune cells and cytokines (LaMarca et al.,
2013; Prins et al., 2009; Santner-Nanan et al., 2009; Wallace et al., 2011). The
amendment of immune balance is considered as key for all pathophysiological
mechanisms associated with Preeclampsia such as reactive ROS production (Mittal et
al., 2014; Dhillion et al., 2012), upregulated expression Endothelin (Granger et al.,
2001b; Granger et al., 2002; LaMarca et al., 2005; LaMarca et al., 2008) and
autoantibodies production by B-cell, which all results in pregnancy hypertension
(Irani et al., 2010; Parrish et al., 2011; Wenzel et al., 2011; Zhou et al., 2008).
2.3.3. Oxidative stress and endothelial dysfunction
In oxidative stress the relative proportion of pro-oxidant species is much higher than
antioxidant species (Myatt and Cui, 2004, Kalyanaraman, 2013). Oxidative stress
results from the imbalance between the intracellular production of (ROS) and
system’s defending ability. Increased production of ROS or a decreased production of
anti-oxidative substances can disturb the balance of oxidants and antioxidants.
Oxidative stress can activate a several biochemical reactions, which can be damaging
for normal human physiology. Normal gestation physiology requires appropriate
placental oxygenation. High fluxes of placental oxygenation increases ROS which
functions as signalling molecules (Kalyanaraman, 2013) which are involved in several
fundamental processes such as replication, fetal growth, cell maturation, proliferation
and gestation maintenance (Multinati et al., 2013). In normal pregnancy ROS are
increased which are necessary for the normal physiology (Yang et al., 2012).
Excessive signalling by ROS is regulated by antioxidant host defence system.
24
As reported previously Preeclampsia is originated from impaired placentation

(Redman and Sargent, 2005). Shallow invasion of trophoblast results in incomplete
vascularization of maternal arteries (Verlohren et al., 2010) and poor blood flow
through placenta. Reduced placental flow creates hypoxic environment, which
favours oxidative stress and consequent affects (Myatt and Webster, 2009). Maternal
system raises the blood pressure to meet the nutrient requirements of the fetus. This
increased blood pressure results in placental reperfusion and consequently reperfusion
injury. Recreated blood flow after reperfusion injury releases inflammatory cytokines.
ROS levels are raised in response to the inflammatory cytokines which eventually
triggers the cell apoptosis pathway (Yiyenoglu et al., 2013).The inflammatory
cytokines are associated with the endothelial cell dysfunction (Myatt and Webster,
2009). Oxidative stress markers like MDA were found significantly higher in cases as
compared to controls (Gowda et al., 2010). The levels of oxidative stress markers
were higher in women with Preeclampsia at gestational age 10-12 weeks, which
suggests the existence of oxidative stress before the onset of clinical symptoms (Genc
et al., 2011).
2.3.4. Genetic predisposition and epigenetic influences
The strong involvement of genetics in the etiopathogenesis of Preeclampsia is evident

from the familial clustering of cases within blood relation and over the generation
(Chappell and Morgan, 2006; Lachmeijer et al., 2002; Wilson et al., 2003). The
genetics behind Preeclampsia becomes more complex as there are two genetic
systems are involved, one maternal and other fetal. The heterogeneity and diversity of
pregnancy hypertensive disorder may be a result of the interaction between maternal,
fetal genes and the environment.
Many studies have reported that, woman having history of Preeclampsia in her first
degree relation (mother, sister or both) then the risk of developing Preeclampsia
increases by 3-5 times (Dukkit and Harrington., 2005; Esplin et al, 2001). The risk
increases 1.5- to 2-fold if father was born to a preeclamptic woman (Esplin et al,
2001; Skaerven et al., 2005), which suggests that predisposition can also be
determined by paternal genes acting through the fetus. A cohort study reported that
25
maternal genes were responsible for up to 35% and fetal genes for up to 20% chances
to develop Preeclampsia (Cnattingius et al., 2004). Candidate gene study and genome
wide studies were done to find the feasible association of genetics and the risk of
developing Preeclampsia. In candidate gene approach a single gene is chosen and its
role in susceptibility of Preeclampsia is studied. Case- control types of study designs
are generally selected for candidate gene studies. For these studies the inclusion
criteria for the patients must be very precise. In candidate gene approach
approximately 70 candidate genes were studied including genes coding for vasoactive
proteins, oxidative stress, lipid metabolism, endothelial injury and immune-genetics
and their role in the Preeclampsia etiopathogenesis was established. (Mutze et al.,
2008). Factor V Leiden (F5), methylenetetrahydrofolate (MTHFR) and prothrombin
(F2) are the most widely studied thrombophilia factors, which are associated with
Preeclampsia, however many studies have also reported contradictory results (Mutze
et al., 2008). A recent study has reported increased risk of Preeclampsia in the
presence of 1691G>A mutation in F5, while MTHFR and F2 has no associations with
Preeclampsia. (Lin et al., 2005) The renin-angiotensin system (RAS) has a vital role
in cardiovascular and renal changes regulation during gestation. Several studies have
associated the RAS with Preeclampsia (Shah et al., 2006). Among all genes of RAS
Angiotensin-converting enzyme, (ACE) and angiotensinogen (AGT) have been
studied extensively in Preeclampsia. Endothelial nitric oxide synthase 3 (eNOS 3) is a
vasodilator and shows reduced activity in Preeclampsia (Wu et al., 2009). eNOS 3
polymorphism had initially been associated with Preeclampsia but was not associated
with increased risk (Medica et al., 2007). Vascular endothelial growth factor (VEGF)
regulates endothelial cell proliferation, migration and vascular permeability. VEGF
polymorphisms 405G>C and 936C>T, were found to be associated with the severity
of Preeclampsia, but could not emerge as risk factors (Banyasz et al., 2006;
Papazoglou et al., 2004). Among immune-genetics TNF-α and interleukin-10 (IL-10)
has been studied. Increased levels of TNF-α contributes to endothelial cell activation,
which leads to maternal symptoms associated with Preeclampsia (LaMarca et al.,
2007). The commonly studied variant of TNF-α is –308G>A located in the promoter
region. This G to A transition contributes to increased levels of TNF-α and an
increased risk for Preeclampsia (Elahi et al., 2009; Saarela et al., 2005). A meta-
26
analysis of 16 studies in which promoter SNP were investigated, could not find any
significant association of TNF-α with Preeclampsia (Bombell and McGuire, 2008).
Preeclamptic placentas show downregulated expression of IL-10 (Markis et al., 2006)
The role of IL-10 variants in Preeclampsia was studied by several groups and
conflicting results were found (Daher et al., 2006; Goddard et al., 2006; Kamali-
Sarvestani et al., 2006). Among the matrix degrading enzyme MMP-9 is extensively
studied. Few studies reported significant role of MMP-9 ‒1562C/T polymorphism in
Pregnancy hypertension (Palei et al., 2010), while in few other studies no association
was found (Gong et al., 2014; Coolman et al., 2007).
Epigenetics refers to the changes in the gene expression without alteration in the DNA
sequence. Epigenetic modification occurs by histone modification by methylation,
acetylation or phosphorylation etc. During pregnancy a women is exposed to several
physiological and social adjustments. Epigenetic modification may play important
role as it could possibly alter the phenotype of placenta (Gheorghe et al., 2010).
Differential expression of miRNA was observed from the microarray genome
profiling of preeclamptic placental tissues (Kleinrouweler et al., 2013). In a case
control study differential methylation pattern was observed (White et al., 2016). A
genome-wide DNA methylation study revealed aberrantly altered DNA methylation
pattern of few genes in preeclamptic placenta (White et al., 2016).
2.3.5. Stage wise model of Preeclampsia
Two-stage model of Preeclampsia was proposed by Redman and Sargent (2005).

Since then the scientists are searching for factor/factors, which connects placental
hypo-perfusion with maternal syndrome. Many scientific groups have suggested that
stage I is not sufficient to cause Preeclampsia in women, therefore the concepts of
“placental preeclampsia” and “maternal preeclampsia” were suggested (Redman and
Sargent, 2005). Inappropriate placentation and maternal intolerance are the causes of
placental preeclampsia. Maternal preeclampsia arises when a mother is predisposed to
systemic inflammation (e.g. chronic hypertension and obesity). Most of the cases are
the combination of both maternal and placental preeclampsia, which is difficult to
separate clinically.
27
Figure 5: Stage wise model of preeclampsia development: Inflammation has a role in the
Preeclampsia etiopathogenesis. The cause of inflammation could be Maternal
immunological intolerance or placental hypo-perfusion (Stage-I). Exaggerated
inflammation favours the condition of oxidative stress and endothelial dysfunction
(Stage-II). The Morbid factors produced because of oxidative stress and endothelial
dysfunction, alone or in conjunction with maternal factors which grounds
inflammation (Obesity, chronic hypertension and genetic factor) can contributes to
maternal syndrome (Stage-III). (Modified: Roberts and Gammill, 2005 and Staff
et al., 2014)
2.4. PREDICTION OF HIGH RISK WOMEN
The quest of dependable and cost-effective screening tests for the prediction of high
risk women for preeclampsia has been the target of research scientists for many years
to improve the maternal and fetal survival rates and minimize the associated adverse
outcomes. Till date, no tests have been found to be effective as screening tests for
preeclampsia (Friedman et al., 2001).This may be attributed to the heterogeneity of
hypertensive disorders in pregnancy, to the different diagnostic criteria and to
differences in outcome measures. Extensive research in last two decades has
identified a list of biophysical and biochemical markers for impaired placentation,
which is considered as the root cause of the Preeclampsia (Wright et al., 2012;
Akolekar et al., 2012). Several studies evaluated the role of combined markers
28
including maternal medical history, UtA PI, MAP, PAPP-A and Free β-hCG at 11-
13weeks for prediction of early onset of Preeclampsia (Wright et al., 2012; Akolekar et
al., 2012). Maternal demographic characters are potential predictors for preeclampsia
when combined with other factors by multivariate analysis (Poon et al., 2010).
History of preeclampsia in previous pregnancy, autoimmune disease, elevated

triglycerides, chronic renal or cardiac disease, excessive weight gain during
pregnancy, interval of two consecutive pregnancies more than ten years and infection
during pregnancy are recognized maternal risk factors for preeclampsia (WHO, 2011;
Magee et al., 2008).
Several candidate markers have been studied, including maternal serum levels of
hCG, uric acid, urinary kallikrein, fibronectin, and urinary calcium, but none of these
were found to be clinically specific and sensitive (Lim and Watkins, 2004). For the
screening of women who are at high risk of developing Preeclampsia, maternal
history, biophysical and biochemical markers are said to be promising (Poon et al.,
2014). Maternal demographic characters like medical and obstetric history (WHO,
2011; Magee et al., 2008) when combined with various factors are reported to be
potentially efficient markers for screening of Preeclampsia (Askie et al., 2007).
Among the biophysical markers, uterine artery Doppler is most assuring. It is non-
invasive method by which uteroplacental circulation can be assessed. Increased
uterine Doppler indicates the impaired implantation (Papageorghiou et al., 2002;
Martin et al., 2001; Plasencia et al., 2007) which is considered as the initiation of
disease development. Among the maternal serum markers, anti-angiogenic markers,
immunological markers, metabolic markers and endocrine markers are reported to be
associated with prediction of Preeclampsia (Petla et al., 2013; Kar, 2014). sFLT-1 and
sEng are antiangiogenic factors which were associated with first trimester prediction
of Preeclampsia (Robinson and Johnson, 2007; Vuorela et al., 2000; Rajakumar et al.,
2005; Levine et al., 2004). sFLT-1 is known to neutralize the effect of PLGF and
VEGF, while sEng affects the production of nitric oxide and vasodilation by
interfering the binding of TGFβ1 to its receptor. The raised maternal serum levels of
sFLT-1 and sEng are associated with the Preeclampsia (Vuorela et al., 2000;
Rajakumar et al., 2005; Levine et al., 2004). sFLT-1 starts increasing in first trimester
as early as 5 weeks (Levine et al., 2004). Serum levels of sFLT-1 and sEng remains
29
elevated throughout the pregnancy which are destined to Preeclampsia as compared to

normal pregnancy (Levine et al., 2004; Robinson and Johnson, 2007 ). sFLT-1 have
shown the 89% sensitivity and 90% specificity for early Preeclampsia, which is much
higher than 55% sensitivity and 58% specificity for late Preeclampsia (Levine et al.,
2006). The ratio of antiangiogenic and proangiogenic i.e. sFLT-1/ PLGF ratio is
considered better predictor of Preeclampsia than measuring them alone (Levine et al.,
2004; Levine et al., 2006). Angiogenesis is the key process in vascular remodelling
during pregnancy and both the splice variants (PLGF and VEGF) of VEGF are key
players. The levels of PLGF and VEGF are decreased in Preeclampsia (Robinson and
Johnson, 2007; Hangmann et al., 2012). Placental protein 13 (PP-13) serves as an
immunological function at feto-maternal interface and vascular remodelling of
maternal arteries (Nicolaides et al., 2006). PP-13 levels are decreased in first trimester
in serum of women who develops Preeclampsia and IUGR (Burger et al., 2004). Low
level of PP-13 is associated with early onset of Preeclampsia but not with the severity
of the disease (Chafetz et al., 2007). PAPP-A is highly glycosylated protein complex,
which cleaves the IGF and makes it available for biological functions (Bersinger et
al., 2009). It has been reported that decreased first trimester plasma levels of PAPP-A
are associated with risk of Preeclampsia (Yaron et al., 2002). However PAPP-A is
known to be more specific for IUGR (Canini et al., 2008). Previous studies revealed
that any markers when used in combination become more specific rather than alone.
On the basis of several reports, few markers were proposed as the candidate markers
for the prediction of Preeclampsia. These markers includes matrix metalloproteinases
(ADAM and MMP-9), inflammation markers (cytokines, CRP, Tumor necrosis factor
receptor-1), Pregnancy markers (PAPP-A, Human chorionic gonadotropin), Fetal
markers (Fetal DNA, Free fetal hemoglobin), endothelial dysfunction (check) marker
(endothelin), tyrosine kinase (Soluble fms-like tyrosine kinase) and genetic markers.
2.5. ROLE OF PLACENTAL BIOMARKERS IN PREECLAMPSIA
The Placenta is considered as the central cause of Preeclampsia Etiopathogenesis.

Human placenta produces several specific proteins, which are found in trace amounts
in the serum. These specific proteins are involved in the programming of trophoblast
invasion. The secretion of these proteins depends upon the hormonal dependent cross
30
talk between placenta and the growing embryo. Poor quality of embryonic signalling
leads to inadequate production of invasion promoting proteins, which consequently
leads to incomplete trophoblast invasion and impaired implantation. Human chorionic
gonadotropin (hCG) is a well-known hormone as placental secretion while Pregnancy
associated plasma protein- A (PAPP-A) is being known from past decade which was
isolated from pregnancy serum 1974. Both PAPP-A and free β-hCG both are secreted
by syncytiotrophoblast.
2.5.1. Role of PAPP-A in Preeclampsia
PAPP-A is serum glycoprotein, which is currently used as maternal serum markers for
10 and 14 weeks screening. PAPP-A is secreted by syncytiotrophoblast and decidua.
PAPP-A is known to increase the bioavailability of insulin like growth factor (IGF),
which is involved in trophoblast invasion and glucose and amino acids transport
through placenta. PAPP-A is also expressed in ovarian granulose cells, and in non-
reproductive tissues, such as fibroblasts, osteoblasts and vascular smooth muscle
cells. PAPP-A is a secreted metalloproteinase encoded by PAPP-A gene, which is
assigned to human chromosome 9q 33.1 (Berends et al., 2007) span above 200 kb of
DNA, and comprises 22 exons (Overgard et al., 2003). PAPP-A belongs to metzincins
superfamily of metalloproteinase, which includes four families of zinc peptidases with
members from both the prokaryotes and eukaryotes –actacins, reprolysins, serralysins
and matrix metalloproteinases (MMPs), also called as matrixins (Boyd et al., 1987).
PAPP-A is known as the first member of a new metzincin family, the pappalysins.
PAPP-A is 400 kDa glycoprotein and produced as a homodimer linked by disulphide
bond. Mature PAPP-A subunit contains 1,547 amino acid residues and 14 putative N-
glycosylation sites (Fialova and Malbohan, 2002; Overgard et al., 1999). Each subunit
of homodimer is formed after processing of 1,627 residue proteins of prepro PAPP-A
(Fialova and Malbohan, 2002; Overgard et al., 1999; Overgard et al., 2001).
Intracellular cleavage of the C-terminal side of PAPP-A polypeptide subsequent to
secretion of active protease, PAPP-A (Fialova and Malbohan, 2002). PAPP-A is
found in free forms or PAPP-A/proMBP (proform of eosinophil major basic protein)
heterotetrameric complex of 500 kDa (Overgard et al., 2003; Qin et al., 2005).
ProMBP in heterodimeric complex is replaced with N- and O-linked glycosylation
31
and single heparin sulfate moiety addition, which is assumed to conquer the PAPP-A
cell-surface binding site in PAPP-A/proMBP heterodimer.
Figure 6: PAPP-A exists in two forms during pregnancy: 1) Free form PAPP-A as a
disulfide-bound homodimer of 200 kDa 2) heterotetramer complex with pro-form
of eosinophil major basic protein (ProMBP) i.e. PAPP-A/ProMBP(Source: Shiefa
et al., 2013)
The molecule of PAPP-A possesses sequence motifs common to metzincin

superfamily of metalloproteinase. PAPP-A molecule has an elongated zinc-binding
motif and strictly conserved methionine residue as a part of superimposable “Met-
turn”, which is supposed to be important for the integrity of the active site. The
catalytic domains of PAPP-A have a core architecture of five ß-strands constituting
one ß-sheet, and three α-helices. Besides these elements, three helices are predicted to
be inserted between five ß-strands. These three helices are not observed in other
members of this family. The active site lies along the edge of an outer strand of the ß-
sheet in a cleft between two half-domains. In addition to the elongated zinc-binding
motif, the C-terminal part of PAPP-A contains approximately five 60-residue motifs
related to the short consensus repeats (SCR). The molecule of PAPP-A contains three
approximately 26-residue motifs related to the lin-notch repeats (LNR) (Fialova and
Malbohan, 2002).
32
PAPP-A is a syncytiotrophoblast secreted metalloproteinase, that enhances the

mitogenic function of the insulin-like growth factor by cleaving the complex of IGF
(Insulin-Like Growth Factors and IGFBP (Insulin-Like Growth Factor binding
protein) (Guibourdenche et al., 2003). Because IGF system is believed to play a
significant role in the trophoblast invasion, so the low concentration of the PAPP-A
might be associated with a higher incidence of the preeclampsia (Poon et al., 2009a).
Figure 7: Proteolytic cleave of IGFBP-IGF complex by PAPP-A. Free IGF involves in

transcriptional activation and amino acid transport
In women, PAPP-A levels are highest during pregnancy, when plasma levels increase
by a factor of about 150 as compared to the non-pregnant state (Poon et al., 2009a).
PAPP-A is most abundant in the peripheral maternal circulation with a mean vascular
content of 250 mg at term. In women with singleton pregnancies, PAPP-A was first
detected in the maternal blood about 28 days post implantation. Serum PAPP-A
concentration increases exponentially with doubling time of 3–4 days during the first
trimester, and then the levels continue to rise throughout pregnancy until delivery. It
can be seen that the concentration rises up at a lesser gradient to 36 weeks after which
the levels increase more steeply right up to term. Maximum levels are attained at
term. Maternal serum hCG level during normal pregnancies is highest at 8 to 10
weeks of gestation, which decline and becomes stable at 18 to 20 weeks of gestation.
PAPP-A and β-hCG are currently being used as markers for screening between 10 and
14 weeks. PAPP-A is predominating IGFBP-4 proteinase in pregnancy serum
33
(Fialova and Malbohan, 2002). It increases the bioavailability of insulin like growth
factor, which in turn mediates trophoblast invasion and modulates transport of glucose
and amino acids through placenta. Although earlier case-control studies did not report
any significant differences in obstetrical outcomes associated with PAPP-A level,
more recent cohort studies describe increased adverse obstetrical outcomes associated
with PAPP-A level below the fifth or tenth centile [< 0.4 MoM (Multiple of median)
or 0.5 MoM (Multiple of median)] (Laursen et al., 2001). Low levels of pregnancy
associated plasma protein-A (PAPP-A) have been previously shown to be associated
with pregnancies that subsequently develop preeclampsia (Spencer et al., 2008; Di
Lorenzo et al., 2012). In chromosomally normal pregnancies there is evidence that
low maternal serum PAPP-A is associated with increased risk for subsequent
development of preeclampsia (Czekierdowski et al., 2008. There are no reports
available describing any adverse obstetrical outcomes with the elevated PAPP-A
levels in the first trimester (Gagnon et al., 2008). There are studies which reported that
PAPP-A in combination with maternal factors, or uterine Doppler is better predictor
for Preeclampsia ( Di Lorenzo et al., 2012) while another report shows no significant
contributions of these factors (Goetzinger et al., 2010). Maternal circulating
concentrations of PAPP-A at 8–14 weeks gestation is significantly predictive of
adverse perinatal outcome in later pregnancy (Smith et al., 2002).
2.5.2. Role of Free β- hCG in Preeclampsia
hCG is 39.5 kDa glycoprotein hormone usually found in blood and urine of pregnant
women. Banerjee et al (2005b) stated that increased levels of hCG in maternal serum
are associated with Down’s syndrome. hCG facilitates various decidual functions
such as endometrium growth and differentiation and fetal functions such as
syncytiotrophoblast growth. hCG has been reported to suppress maternal immune
system during pregnancy, accent uterine morphology and synchronize the signal
transduction at feto-maternal interface (Banerjee and Fazleabas, 2011).
hCG hormone is a heterodimer formed by two subunits α and β which are linked non-
covalently. This hormone is produced by syntiotrophoblast. The α subunits has 92
amino acid residues linked together by five disulfide bonds. The α subunits have
similar structure to other three glycoprotein hormone named LH, FSH, and TSH. The
34
β subunit has 145 amino acid residues linked together by six disulfide bonds.
Functionally β subunit is dissimilar from the one present in intact hCG. The β subunit
has two N-linked oligosaccharide units and four O-linked oligosaccharide units. N-
linked oligosaccharide units are coupled to amino acid residues at positions 13 and 30
(Stenman et al., 2006). O-linked oligosaccharide units are sited in the exclusive
proline-serine-rich C-terminal flange after amino acid residues 122 to145. In α
subunits two N-linked oligosaccharide units are linked to amino acid residues 52 and
78 (Stenman et al., 2006). Two subunits α and β are encoded by different genes
located on different chromosomes. The gene encoding for α subunits is located on
chromosome 6 and for β subunit located on chromosome 19 (Reis et al., 2002).
Distinct messenger RNAs (mRNAs) molecules are transcribed and translated. The
two subunits intuitively combine in endoplasmic reticulum. Maternal serum contains
hCG molecules in five forms: intact hCG, nicked hCG; free α subunit; free β subunit;
and nicked free β subunit (Montagnana et al., 2011).
Figure 8: Structure of intact hCG and free β-hCG:α and β subunits are linked by
disulphide bond (upper section); N-linked glycosylation at two amino acid
residues in free β Subunits (Lower section) (Modified: Shiefa et al., 2013)
35
Serum hCG levels are maximum at 8–10 weeks and then drops to reach a plateau at
18–20 weeks of gestation. It has been suggested that β-hCG obstructs with the
growth-inhibiting effect of transforming growth factor-β (TGF-β), platelet-derived
growth factor-β and nerve growth factor (Butler and Iles, 2004). A study has reported
that Downs’s syndrome trophoblasts indicates as striking increase in mRNA level of
β-hCG with a smaller increase in α-hCG mRNA levels which is explanatory for
elevated hCG levels in maternal serum. The amount of free β-hCG circulates in
maternal serum is equivalent to 0.3–4 % of the total hCG (Reis et al., 2002). The free
β-subunit can arise from direct synthesis by syncytiotrophoblast, cleaving of intact
hCG into α and β subunits, and by nicking of hCG molecule by enzymes produced by
macrophage or neutrophil (Reis et al., 2002).
Low free β -hCG levels are associated with early pregnancy loss. Increased β -hCG
levels in second-trimester have been associated with a number of adverse obstetric
outcomes Preeclampsia attributable to placental dysfunction (Dugoff, 2010). There is
a conflicting data regarding the levels of the free β- hCG and Preeclampsia. A recent
report shows that free β- hCG can provide possible screening for Preeclampsia in first
trimester (Asvold et al., 2014) while another report says that β- hCG has no
significant role in preeclampsia (Goetzinger et al., 2010). Elevated β- hCG levels in
second trimester are associated with subsequent development of Preeclampsia and
other adverse obstetric outcomes (Wald and Morris, 2001) while other studies have
shown no difference in β- hCG levels during first trimester (Sebire et al., 2000). Kaur
et al (2012) reported that increased level of β- hCG at 13-20 weeks can predict
preeclampsia with positive predictive value of 83.33%.However Asvold et al (2014)
reported contrasting results. He found that β- hCG levels during first trimester shares
an inverse relation with preeclampsia development i.e. low level of free β- hCG is
associated with subsequent development of preeclampsia. Considerable evidence
suggests an association between serum hCG levels and preeclampsia (Banerjee et al.,
2005b). Correlation of serum level of β-hCG with the severity of preeclampsia might
reflect a different trophoblastic secretory response of this disease.
36
2.6. ROLE OF CYTOKINES IN PREECLAMPSIA
Small, non-structural proteins with molecular weights ranging from 8 to 40KDa are
Cytokines which regulate immune responses to infections, inflammation and trauma.
Pro-inflammatory cytokines (TNF- α and IFN-γ) worsen the disease, whereas others
group of cytokines acts antagonistically to reduce inflammation and promote healing
are anti-inflammatory (IL-4, IL-10, and IL-13). The trophoblast invasion is regulated
by several cytokines produced by several cells of immune and non-immune origin,
such as leukocytes including NK cells, the trophoblast and glandular endothelium at
the maternal- fetal interface. The current hypothesis of the aetiology of preeclampsia
revolves around two factors: 1) intolerance of maternal immune system and 2)
defective trophoblast invasion. The activation of the adaptive immune response is
characterized according to the phenomenon of polarized cytokine secretion by T-
helper (Th) cells. These are primarily divided into two groups: Th1 and Th2 cells. Th1
cells produce inflammatory cytokines like TNF- α and IFN- γ whereas Th2 cells
produces IL-4, IL-5 and IL-9, which are anti- inflammatory in nature (Redman and
Sargent, 2008).
Chronic immune activation is associated with Preeclampsia. Immune activation leads

to an augmented production of inflammatory cytokines by pro-inflammatory T-cells.
The production of regulatory and anti-inflammatory cytokines is reduced, which
further promotes the inflammatory state in preeclamptic pregnancy (Bennett et al.,
1998; Hanna et al., 2000 Chatterjee et al., 2015). This imbalance between anti-
inflammatory and pro-inflammatory cytokines is a result of placental ischemia, which
is followed by impaired implantation of trophoblast. This imbalance further shift
towards pro-inflammatory cytokines and the condition worsens with the increasing
gestation (LaMarca, 2010). Anti-inflammatory cytokines like interleukin (IL)-10 and
IL-4 which controls the immune response, play important roles in a normal and
successful pregnancy by balancing immune system (Chatterjee et al., 2015).
Activated Th1 and Th17 cells secrete pro-inflammatory cytokines such as TNF-α, IL-
6 and IL-17 during immunological encounters, which creates cytotoxic and
inflammatory responses (Redman and Sargent, 2010; Gadonski et al., 2006; Keiser et
al., 2009; LaMarca and Zhou, 2011). Increased levels of TNF-α and IL-6 were found
37
in the maternal blood circulation and trophoblastic cells of the placenta, while IL-10
and IL-4 were decreased (Keiser et al., 2009; Arriaga-Pizano et al., 2005; Hennessy et
al., 1999). This imbalance of pro-inflammatory and anti-inflammatory cytokines
promotes chronic placental and peripheral inflammation, which plays a role in further
etiopathogenesis of Preeclampsia.
2.6.1. Role of TNF-α in Preeclampsia

TNF- α is a multifunctional pro-inflammatory cytokine which is a member of tumor
necrosis factor (TNF) superfamily and encoded by TNFA gene which is located on
chromosome 6 at 6p21.3 region. TNFα was first recognized as a cytokine in the
1970s. Rusten (1994) stated that TNF-α has ability to suppress cell proliferation.
TNF-α is synthesized as a pro-TNF-α which is a membrane-bound protein. Pro TNF-α
is released by TNF-converting enzyme (TACE)- mediated cleavage. TNF- α is a
homotrimer, where each monomer has 212 amino acid residues. TNF- α is type-II
transmembrane protein molecule. Three monomer units are joined about 3 fold around
axis to form bell shaped trimer. This type of monomer arrangement is specific for
TNF family proteins, but it shares homology with some viral coat proteins. TNF-α
executes its biological function through cell membrane bound receptors, TNFRs. At
the time of decidualization TNF-α was found to be expressed by stromal cells and
macrophages, uNK (uterine natural killer cells) and epithelial cells (Haider and
Knofler, 2009).
TNF- α is involved in the regulation of a wide spectrum of biological processes,

including cell proliferation, differentiation, apoptosis, lipid metabolism and
coagulation (Sriram and O’Callaghan, 2007). This cytokine has been implicated in a
variety of diseases, including autoimmune diseases (Taylor et al., 2000), insulin
resistance (Swaroop et. al., 2012), and cancer (Wang et al., 2013).
In preeclamptic women increased levels of TNF-α in conjunction with other cytokine

is known to contribute to endothelial dysfunction, which is a hallmark of
Preeclampsia (Benyo et al., 2001; Gu et al., 2008). Endothelial dysfunction is
characterized by increased level of adhesion molecules and endothelial cell
penetrability (LaMarca et al., 2005). TNF-α downregulates the expression of nitric
oxide synthase mRNA and increases the production of Endothelin by increasing the
38
expression of preproendothelin-1 mRNA (Foschi et al., 2001; Yan et al., 2008).

Endothelin levels were found to be increased by 2–3-fold in Preeclamptic women.
The expression of preproendothelin-1, which is a precursor of Endothelin increased in
preeclamptic in women as compared to normotensive pregnant women (LaMarca et
al., 2005).
The elevated concentration of TNF-α in the preeclamptic women (Vahid et al., 2009)
is known to promote apoptosis and leakage of blood vessels leading to systemic
endothelial activation and subsequently to the development of the clinical signs
associated with the preeclampsia (Senhaeve et al., 2006).
In conjunction with an over-expression and secretion of TNF-α in the placenta and

circulation in preeclampsia an enhanced expression of IL-1 is also reported (Senhaeve
et al., 2006). IL-1 and TNF-α both promote structure and functional changes in the
endothelial cells, including oxidative stress, activation of the complement cascade,
secretion of the vasoconstrictor, micro thrombosis and infraction and elevated
thromboxane levels.
TNF-α has also been shown to elevate leptin protein level as a phenomenon
associated with preeclampsia. Microarray analysis of differentially expressed gene in
placental tissue of the preeclampsia revealed that one of the most up-regulated
transcripts in preeclampsia tissue was the obese leptin gene (Reimer et al., 2002).
TNF-α seems to be involved in the pathophysiologic mechanisms leading to the
clinical signs of the preeclampsia. Fishelson et al., 2001; Gibert et al., 2008).Thus,
TNF-α is a major contributor to many of the local and systemic changes that
characterizes preeclampsia.
The plasma levels of TNF-α during first trimester are found to be elevated in the
preeclamptic cases as compared to controls, but these levels are not useful as a
specific marker for prediction of preeclampsia in the first and second trimester (Serin
et al., 2002). Very few studies are available which demonstrates the first trimester
levels of TNF-α in preeclamptic women. Gupta and Chari (2015) found no significant
difference in serum levels of TNF- α in first trimester in preeclamptic women as
compared to normotensive (Gupta and Chari, 2015). Similar findings were reported
39
by Serin et al (2002) who observed no significant change in levels of TNF-α between

the gestational age of 9-17 weeks. Hamai et al (1997) found contrasting results with
statically significant elevation in serum level of TNF- α during first trimester in
preeclamptic women.
2.6.2. Role of IFN-γ in Preeclampsia
IFN-γ is a dimerized soluble cytokine that is the only member of the type II class of
interferons and is encoded by the INFG gene (Gray and Goeddel, 1982; Naylor et al.,
1983). IFN-γ or type II interferon is a cytokine that is critical for innate and adaptive
immunity against viral and intracellular bacterial infections and for tumor control.
Aberrant IFN-γ expression is associated with a number of auto-inflammatory and
autoimmune diseases. The importance of IFN-γ in the immune system stems in part
from its ability to inhibit viral replication directly and most importantly from its
immune stimulatory and immunomodulatory effects. IFN-γ is produced
predominantly by natural killer (NK) and natural killer T (NKT) cells as part of the
innate immune response, and by CD4 and CD8 cytotoxic T lymphocyte (CTL)
effector T cells once antigen-specific immunity develops.
IFN- γ released by activated T-Cells activates the specialized uNK cells which
possess regulatory properties for physiological trophoblast invasion in the decidua.
However, excessive amounts of IFN- γ in conjunction with TNF-α and IL-1 can lead
to apoptosis of the trophoblast. In an inflammatory environment macrophages secret
high level of IL-12 that stimulate IFN- γ secretion by NK cells, thereby inhibiting the
angiogenesis. It has been evidenced by Hu et al. (2007) that IFN- γ inhibits the migration of
cytotrophoblast in explant cultures. There are several studies showing the increased levels of
the IFN- γ in the third trimester of pregnancies with preeclampsia (Arriaga-Pizano et al.,
2005; Banerjee et al., 2005a; Murphy et al., 2009).
As far as our knowledge is concerned there is no study available reporting the first
trimester serum concentration of the IFN- γ for prediction of preeclampsia or any
other obstetric complication. Tangeras et al. (2015) reported a different Cytokine
profile in First trimester of women who later developed pregnancy hypertension
disorders as compared to controls.
40
2.7. ENDOTHELIAL DYSFUNCTION IN PREECLAMPSIA:
The blood vessels have a single cell lining membrane on the inner wall called
endothelium. Originally endothelium was known to have a function of barrier only
later by the discovery of endothelium derived relaxing factors (EDRF) by Furchgott
and Zawadzki (1980) (85.1) a window of new possibility in vascular biology was
opened. Placenta is the origin of Preeclampsia but the endothelium is the tissue which
is most affected. Altered state of endothelial cell differentiation is known as
endothelial cell dysfunction or activation, which could be a result of cytokine
stimulation. Numbers of studies are available to support a major role of endothelial
dysfunction in vasospasm and high vascular reactivity, which are the final and
common pathway of several pathophysiologic mechanisms of preeclampsia (Roberts
and Lain, 2002; Taylor et al., 2000; Walsh, 1998).
Endothelial dysfunction in the central contributor to the maternal syndrome including

hypertension, proteinuria, and edema in Preeclampsia (Roberts and Cooper, 2001).
Previous studies provide evidence that endothelial dysfunction follows abnormal
placentation, which causes placental ischemia and release of placental factors that
damage the maternal vascular endothelium (Roberts and Cooper, 2001; Granger et al.,
2001b). There are studies that indicate central role of oxidative stress in endothelial
dysfunction in Preeclampsia (Roberts and Hubel, 2009; Walsh, 1998). A Recent study
has shown that antioxidant vitamins can improve biochemical markers of endothelial
activation thus minimizes the development of Preeclampsia in high-risk women
(Chappell et al., 1999). Increased peroxynitrite production by lipid peroxidation in
preeclamptic placentas (Vanderlelie et al., 2005; Gupta et al., 2005) led to a theory
that placenta is the principal cause of oxidative stress. Endothelial dysfunction was
found to be more severe in women who had Preeclampsia in their previous pregnancy
and was not attributed to obesity, hypertension and metabolic syndrome which are
considered as potential factors of vascular function. The clinical signs of Preeclampsia
imitate widespread endothelial dysfunction, with vasoconstriction and end organ
ischemia. Systemic hypertension, renal, hepatic, and cerebral vascular pathology are
hallmarks of severe Preeclampsia but Taylor et al. (2009) suggested the evidence of
endothelial dysfunction as the principal focus of the disease. They suggested that
41
endothelial activation and dysfunction are result of inappropriate vasoconstriction.

This state has tendency toward a hypercoagulable state. These investigators also
suggested that endothelial dysfunction may be articulated by the reformed synthesis
and release of endothelial cell products (Taylor et al., 2009).
2.7.1. Role of Endothelin and Nitric oxide in preeclampsia
Endothelium produces factors like nitric oxide (Furchgott et al., 1980) prostacyclin
and endothelin (Yang et al., 1997). ET is 21 amino acid long vasoconstrictor found in
three isoforms Endothelin, ET-2 and ET-3. Endothelin is the primary isoform
produced by endothelium. ET execute its function by heptahelical G- protein coupled
receptors ETA and ETB, which are distributed on different tissues suggesting the
multifactorial role of endothelin. Endothelin produced by the basal endothelium and
induces vasoconstriction of smooth muscle by ETA. Endothelin activates neighboring
endothelial cells in autocrine and paracrine manner to produce vasodilators nitric
oxide and prostacyclin by ETB. These findings suggested that Endothelin raises blood
pressure by ETA and reduces blood pressure by ETB.
Nitric oxide is a biological signaling molecule synthesized from L-arginine by nitric

oxide synthases. Nitric oxide is produced in different types of cells and regulates of
several physiological and pathological processes, such as inflammation and
metabolism. Nitric oxide synthase found in three isoforms: neuronal nitric oxide,
endothelial nitric oxide, and inducible nitric oxide (Moshage, 1997). Nitric oxide is
highly unstable; therefore measurement of nitrate and nitrite is used as an index of
nitric oxide production and nitric oxide enzyme activity (Baylic et al., 1998). During
normal pregnancy widespread vasodilation occurs and vascular resistance is reduced.
This extensive vasodilation is associated with increased serum nitrate levels. Raised
systemic arterial pressure and increased vasoconstriction could result due to lower
production of vasodilators (Endemann and Schiffrin, 2004; Gupta et al., 2003). It is
known that both nitric oxide production and response to nitric oxide increase in
normal pregnancy, and it is thought that this increase plays a role in many
physiological mechanisms, which provide continuation of the pregnancy.
42
Nitric oxide production increases with gestational age during normal pregnancy and
decreases in preeclampsia. Hata et al. (1999) reported negative correlation of nitric
oxide production and gestational age. He stated that nitric oxide production decreases
with the advancement of gestational age. The serum level of nitric oxide is reduced in
preeclampsia when compared to the controls (Choi et al., 2002). Lowe (2000) and
Curtis et al. (2009) suggested that nitric oxide does not play a role in etiopathogenesis
of preeclampsia. These observations suggest that the status of nitric oxide
biosynthesis in women during normal pregnancy is still controversial.
Endothelin and nitric oxide are potent vasoconstrictor and vasodilator respectively,
and both are produced by endothelium. The production of endothelin and nitric oxide
depends on the condition of endothelium. The balance between endothelin and nitric
oxide maintains the homeostasis of the blood vessels. During endothelial dysfunction
the production of nitric oxide is downregulated while endothelin is upregulated, which
causes the disturbance of blood vessel homeostasis and promotes vascular disease like
hypertension (Sakurai and Sawamura, 2003). Studies have shown that damage of
intact structure of endothelial by hypertension and inflammatory factors can promote
endothelin synthesis, which enters the blood circulation and results in increased
resistance of blood vessels. At the same time instant blood pressure is elevated by
aldosterone and angiotensin, which subsequently speed up injury of endothelial cells
(Herse and LaMarca, 2013; Perl et al., 2010; Seremak-Mrozikiewicz et al., 2011).
Nitric oxide is a vasodilator, which relaxes smooth muscle, obstruct adhesion,
accumulation of platelet and anticoagulation. Studies have found that increased level
of nitric oxide could effectively maintain the status of blood flow in pregnancy
hypertension so that vascular relaxation of placenta is maintained so that fetus could
grow (Zhang et al., 2013)
It has been suggested that the production of ROS induces endothelial dysfunction.
Due to shallow invasion of trophoblast maternal spiral arteries do not undergo
complete modification to low resistant vessels and could not compensate the oxygen
requirements of the growing fetus, which creates the hypoxic environment. Maternal
system upsurges the blood pressure to accommodate the oxygen requirements of the
fetus. This re-oxygenation or reperfusion results in ROS generation, which exceeds
43
the level of compensating antioxidants. Hypoxia / re-oxygenation cause endothelial

cell injury and inflammation (De Pascali et al., 2014).
2.8. ROLE OF MATRIX METALLOPROTEINASE-9 (MMP-9) IN PREECLAMPSIA
Matrix metalloproteinases (MMPs) also called matrixins are the extracellular matrix
(ECM) degrading enzymes belong to the family of zinc endopeptidases collectively
referred to as metzincins. The metzincins are subdivided into four multigene families:
seralysins, astacins, ADAMs / admalysins, and MMPs. Matrix metalloproteinase -9 is
encoded by MMP-9 gene, located on the 20th chromosome at q13.12 regions.
The MMP family is comprised of more than 20 related zinc-dependent enzymes that
share common functional domains. The metzincin superfamily is distinguished by a
highly conserved motif containing three histidines that bind to zinc at the catalytic site
and a conserved methionine that sits beneath the active site (Bode and Maskos, 2003).
MMPs shows extensive ability to degrade extracellular matrix (ECM) proteins such as
collagen, laminin, fibronectin, vitronectin, aggrecan, enactin, tenascin, elastin, and
proteoglycans (Woosner and Nagase, 2000). Other than ECM degradation, MMPs have a
myriad of other important functions that may be independent of proteolytic activity
(Overall et al., 2002). All MMPs are synthesized in the latent form (Zymogen). They are
secreted as pro-enzymes and require extracellular activation. Matrixins participate in
many normal biological processes e.g. embryonic development, blastocyst implantation,
organ morphogenesis, nerve growth, ovulation, cervical dilatation, postpartum uterine
involution, endometrial cycling, hair follicle cycling, bone remodelling, wound healing,
angiogenesis, apoptosis, etc. and pathological processes e.g. arthritis, cancer,
cardiovascular disease, nephritis, neurological disease, breakdown of blood brain barrier,
periodontal disease, skin ulceration, gastric ulcer, corneal ulceration, liver fibrosis,
emphysema, fibrotic lung disease, etc. (Parks et al., 2004). Although the main function of
matrixins is the removal of ECM during tissue resorption and progression of many
diseases, it is notable that MMPs also alter biological functions of ECM macromolecules
by specific proteolysis.
Vascular remodelling and trophoblast invasion are the main event for successful
embryo implantation. Vascular remodelling is accomplished by reorganization of
44
extracellular matrix (ECM) and angiogenesis (Bonnans et al., 2014). During vascular
remodelling in early phases of pregnancy, pre-existing arteries are modified and
reformed to compensate the blood flow required for growing fetus. Development of
the placenta starts with the invasion and migration of trophoblast cells into the
maternal tissue to establish connection with the maternal circulation.
Trophoblast invasion is achieved by degradation of ECM proteins mainly by MMPs.

The activity of MMPs is strictly regulated by tissue inhibitors of metalloproteinases
(TIMP) (Woessnser and Nagase, 2000). MMP-2 and MMP-9 are known to degrade
collagen and ECM at the placentation site (Ishaka et al., 2003; Woosner and Nagase,
2000). Among all MMPs, MMP-9 and MMP-2 are most important ECM degrading
proteinases during pregnancy (Page-McCaw et al., 2007). Both MMP-2 and MMP-9
shows dissimilar expression during first trimester. MMP-2 is expressed during early
first trimester (6-8 weeks) while MMP-9 expression is overriding in late first trimester
(9–12 weeks) (Staun-Ram et al., 2004; Xu et al., 2000). At the site of implantation,
MMP-9 is extensively expressed by trophoblasts. MMP-9 inhibition decreases
trophoblast migration and ECM degradation (Woosner and Nagase, 2000).
Salamonsen et al (1999) reported a significant role of MMP-9 in early implantation in
mouse blastocyst. Any kind of amendment in the expression of the MMP-9 gene may
lead to the impaired implantation of trophoblast, which is considered as primary cause
of Preeclampsia. Previous studies provide conflicting data for the MMP-9 activity in
Preeclampsia etiopathogenesis. Maternal serum concentration of MMP-9 at 11+0 −
13+6 weeks gestation in pregnancies which subsequently developed Preeclampsia and
results in preterm delivery (Poon et al., 2009c). Karampas et al. (2014) found no
significant association of MMP-9 levels with Preeclampsia in first trimester. Kolben
et al. (1996) reported no significant variation in plasma levels of MMP-9 in
preeclamptic women when compared with normotensive antenatal women. Plasma
level of MMP-9 inhibitor TIMP was elevated in Preeclampsia, but MMP-9/TIMP-1
ratios was not significantly changed (Palei et al., 2008). Similar findings were also
reported by Montagnana et al. (2009) who found no variations in serum MMP-9 and
TIMP-1 in Preeclampsia. A study has been reported positive correlation of increased
level of MMP-9 with TNF-R1, which suggests the presence of an underlying
45
inflammatory process, and can be used as a parameter for the prediction of the
preeclampsia. MMP-9 has been studied in reference to Preeclampsia and the data
observer from previous studies.
MMPs expression was also studied by placental bed biopsies. Huisman et al.
evaluated the expression of MMP-2 and MMP-9 by placental bed biopsies in first
trimester of gestation. They found no significant change in MMP-2 and MMP-9
expression (Huisman et al., 2004). Kolben et al (1996) and Shokry et al (2009)
reported reduced MMP-9 levels in preeclamptic placental tissues as compared to the
normal placentas. Romanowicz and Galewska (2010) found reduced expression of
MMP-9 levels in umbilical cord tissues of preeclamptic placenta.
Abnormal production of MMP-9, may result from functional genetic polymorphisms

in the promoter region of MMP-9 gene, which are known to change MMP-9
expression (Palei et al., 2010). Another study suggested that the altered expression
profile of the MMP-9 gene in preeclampsia might be associated with the methylation
status of CpG sites in the promoter region (Wang et al., 2010).
2.9. GENETIC POLYMORPHISM OF MMP-9 GENE
Existence of two or more type of genotypes in a given population is genetic

polymorphism. SNP is the most common type of genetic polymorphism. If a genotype
is found 1 in 100, it is considered as genetic polymorphism.
MMPs genetic polymorphisms are being increasingly recognized in the context

angiogenesis. Approximately 10 million SNP are in human genome, small part of
which is functionally active. Functionally active SNPs are present in the promoter
region of the gene. Literature shows that mmp-9 promoter region SNPs are related to
the diseases (Langers et al., 2011). Under normal physiological environment, genes
expression is tightly regulated at transcription levels, however in abnormal
physiological state the expression may be aberrant (Libermann and Zerbini, 2006).
The human MMP9 gene is mapped to the chromosome region 20q11.2–q13.1 and
several polymorphisms of this gene were identified. The −1562 C/T polymorphism
46
(rs3918242) was shown to exert a functional effect on gene transcription. This single
nucleotide polymorphism (SNP) at −1562 bp is due to a C to T substitution (−1562
C/T), which results in the loss of binding of a nuclear protein to this region and an
increase in transcriptional activity in macrophages. In these cells, the C/C genotype
leads to a low promoter activity whereas the C/T and T/T genotypes result in high
transcriptional activity (Zhang et al., 1999).
Figure 9: Schematic representation of Human chromosome 20. Location of MMP-9 gene

in 20 q 13.1 region. (Source: Image adapted and Modified from UCSC Genome
Browser (http://genome.ucsc.edu/ cgi-bin/hgGateway)
MMP-9 ‒1562 C/T genetic polymorphism is detected by PCR-RFLP technique and

amplification product obtained is 436 bp (Singh et al., 2012). However, the
amplification product length reported by El Samanoudy et al (2014) was 435bp. The
amplification product is than digested with SphI enzyme. The genomic source of SphI
is Streptomyces phaeochromogenes (Guthrie et al., 1996). SphI identifies
hexanucleotide sequence 5’GCATGC and slices after the second guanine to produce a
4-base 3' overhang (Fuchs et al., 1980).
MMP-9 activities imbalance has been known to affect cardiovascular system,

counting pregnancy hypertensive disorder (Kolben et al., 1996; Palei et al., 2008;
Montagnana et al., 2009; Narumiya et al., 2001; Tayebjee et al., 2004). MMP-9 may
intricate in maternal and fetal arterial remodelling (Ishaka et al., 2003; Kelly et al.,
2003) and in the vascular tone regulation (Chow et al., 2007; Jeyabalan et al., 2007) .
Studies are available which indicatesMMP-9 role in endothelial dysfunction in
47
Preeclampsia patients, by interacting with oxidative stress and inflammatory factors

(Sankaralingam et al., 2006). Genetic polymorphisms of MMP-9 gene influences
MMP-9 transcription. MMP-9 gene has two functional polymorphisms -1562C/T
(single-nucleotide polymorphism) and -90(CA)1325 (microsatellite). In vitro studies
revealed that ‘C’ to ‘T’ replacement at ‒1562 position upsurges MMP-9 expression
(Zhang et al., 1999). However, (CA) 14 allele downregulates the MMP-9 expression
by 50% as compared to (CA) 21allele (Peters et al., 1999; Shimajiri et al., 1999).
Both of these functional genetic variations have been linked to pregnancy
hypertensive disorder Preeclampsia and Gestational hypertension (Coolman et al.,
2007; Palei et al., 2010). These reports signify the role of MMP-9 genetic deviations
in predisposition to pregnancy hypertensive disorders. However, the effect of MMP-9
polymorphisms on MMP-9 serum levels in pregnancy hypertension has not been
studied in reference to Indian populations.
Previous reports have associated MMP-9 −1562C/T polymorphism with

cardiovascular diseases (Bateman et al., 2012; Lelongt et al., 2001), hypertension
(Plaks et al., 2013) and cancer (Rollin et al., 2007; Sfar et al., 2008). Events during
trophoblast invasion are similar to tumorigenesis but trophoblast invasion is regulated
at various checkpoints (Soundararajan and Rao, 2004). The two C and T have
different impact on MMP-9 expression in various types of cancers. Sfar et al (2008)
reported, C allele is associated with a reduced risk of prostate cancer while T allele
upsurges prostate cancer risk. Rollin et al (2007) reported C and T allele impact in
lung cancer. C allele is associated with higher risk of squamous cell carcinoma,
endometrial carcinoma and colorectal cancer (Sugimoto et al., 2006).
The MMP-9 ‒1562C/T polymorphism has been studied in reference to other

Pregnancy complications such as intrauterine growth restriction and recurrent early
pregnancy loss (Gremlich et al., 2007; Singh et al., 2012). MMP-9 ‒1562C/T
polymorphism is associated with high risk of Preeclampsia (Rahimi et al., 2013).
Different results were reported by Palei et al (2010), who found that MMP-9 ‒
1562C/T polymorphism is associated with Gestational hypertension but not with
Preeclampsia. Gong et al (2014) found no significant association of that MMP-9 ‒
1562C/T polymorphism with Preeclampsia.
48
Genetic reports have also been divergent regarding the association of MMP-2 and
MMP-9 polymorphisms with preeclampsia. Coolman et al (2007) observed a reduced
prevalence of the rare T allele of the MMP-9 −1562C/T polymorphism in
preeclampsia (Coolman et al., 2007). Intriguingly, in vitro studies have showed that
the “C” to “T” substitution at −1562 position of the MMP-9 promoter increases
MMP-9 gene expression (Zhang et al., 1999). Therefore, the lower frequency of the
‒1562 T allele in preeclamptic patients suggests that they may have decreased MMP-
9 levels, which might predispose them to maladaptation of the spiral arteries and
decreased degradation of the decidua. However, MMP-9 polymorphisms were not
linked to preeclampsia (Fraser et al., 2008; Palei et al., 2010).
49

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Review of Literature

2.4 Prediction of high risk women

2.6 Role of cytokines in preeclampsia

2.7 Endothelial dysfunction in preeclampsia

Preeclampsia is pregnancy associated disorder, which is initially identifiable by new-

Preeclampsia is a disorder, which is known to affect multiple organs of maternal

2.1. MATERNAL AND FETAL OUTCOME

Hence, in preeclamptic pregnancies, the obstetricians have to balance the needs of

2.2. RISK FACTORS

Table 1: Maternal risk factors for the development of preeclampsia

Factors Risk (Odds Ratio)

BMI >35 kg/m2 2: 1

Preeclampsia in a previous pregnancy 7: 1

Family history of preeclampsia 3: 1

According to WHO (World’s Health Organization) 20% of preterm birth reported

predisposed to gestational complications like Preeclampsia and gestational diabetes

The classification of the pregnancy hypertension disorder is provided by the NHBPEP

2. Preeclampsia and Eclampsia Syndrome

3. Preeclampsia syndrome superimposed on the chronic hypertension

Gestational hypertension → Formerly termed as the pregnancy induced

Preeclampsia → Preeclampsia is characterized by the hypertension and significant

Chronic hypertension→ Hypertension diagnosed before 20th week of gestation and

Preeclampsia syndrome superimposed on the chronic hypertension→ New onset

Diagnosis of the pregnancy hypertensive disorders:

Gestational hypertension • New onset of hypertension (140/90mmHg)

Preeclampsia Mild Preeclampsia:

2.3. ETIOPATHOGENESIS OF PREECLAMPSIA

and prospective follow up of eclamptic women for 43 years. He is credited as an

De Sauvages in 1739 gave his view on the causation of Preeclampsia. He

Figure 1: Two-stage model of Preeclampsia: Preeclampsia is initiated by Impaired

• Placental implantation with abnormal trophoblastic invasion of uterine vessel.

2.3.1. Placental implantation with abnormal trophoblastic invasion of uterine vessel

The placental development is initialized on implantation of the blastocyst into

Successful embryo implantation is consequent result of parallel and synchronised

Figure 2: Embryonic signal produced at feto-maternal interface stimulates morphological

Figure 3: Abnormal Spiral artery remodelling and placentation in preeclamptic

2.3.2. Maternal maladaptive immunological tolerance

Figure 4: Trophoblast invasion regulation by paracrine factors: The signal exchange

Specific mechanisms which promote development of Preeclampsia are yet to be

In contrast to normal pregnancies, preeclamptic pregnancies are associated with

2.3.3. Oxidative stress and endothelial dysfunction

As reported previously Preeclampsia is originated from impaired placentation

2.3.4. Genetic predisposition and epigenetic influences

The strong involvement of genetics in the etiopathogenesis of Preeclampsia is evident

2.3.5. Stage wise model of Preeclampsia

Two-stage model of Preeclampsia was proposed by Redman and Sargent (2005).

2.4. PREDICTION OF HIGH RISK WOMEN

History of preeclampsia in previous pregnancy, autoimmune disease, elevated

elevated throughout the pregnancy which are destined to Preeclampsia as compared to

2.5. ROLE OF PLACENTAL BIOMARKERS IN PREECLAMPSIA

The Placenta is considered as the central cause of Preeclampsia Etiopathogenesis.

2.5.1. Role of PAPP-A in Preeclampsia

The molecule of PAPP-A possesses sequence motifs common to metzincin

PAPP-A is a syncytiotrophoblast secreted metalloproteinase, that enhances the

Figure 7: Proteolytic cleave of IGFBP-IGF complex by PAPP-A. Free IGF involves in

2.5.2. Role of Free β- hCG in Preeclampsia

2.6. ROLE OF CYTOKINES IN PREECLAMPSIA

Chronic immune activation is associated with Preeclampsia. Immune activation leads