1.0 recurrent pregnancy loss - shodhganga : a reservoir of...

Introduction

A study on genetic polymorphisms associated with unexplained recurrent pregnancy

loss in South Indian Population 1

1.0 Recurrent Pregnancy Loss

Pregnancy is a complex, heterogenous, biological phenomenon in which the embryo

develops into a fetus within the female uterus. The entire process is a vital

immunological paradox, where the semiallogeneic fetus survives by evading maternal

immune recognition and is delivered after the completion of the gestational period.

The duration of gestation is conventionally divided into three trimesters of roughly

three months each. However, due to various etiological factors, the growing embryo

that is unable to survive is expelled from the pregnant mother at different gestational

ages and this is referred to as pregnancy loss or abortion. Pregnancy loss occurs in 10

to 15% of all pregnancies, of which 1-2% are recurrent (1). Recurrent pregnancy loss

(RPL) was initially defined as the loss of three or more clinically recognized

pregnancies spontaneously during early gestation. However, the modern definition

refers to the spontaneous loss of two or more consecutive pregnancies before twenty

weeks of gestation (2). The World Health Organization (WHO) has defined

miscarriage as the loss of a fetus weighing ≤500g, which would normally be at 20-22

complete weeks of gestation (3).

Etiology of recurrent pregnancy loss is among the most studied, yet unresolved issue

in modern gynecology. Among the various proposed etiological factors, abnormal

parental karyotype, antiphospholipid syndrome and uterine anatomic abnormalities

were reported in about 50% of the patients; however, in remaining 50%, the cause is

unknown (4, 5). RPL of unknown etiology provides a fundamental insight into the

processes of embryogenesis and implantation. Epidemiological studies have

suggested that the condition might be multifactorial with a possible genetic

predisposition and environmental factors in its pathogenesis (6, 7). A better

understanding of the role of various gene-gene and gene-environment interactions will

enable identification of high-risk individuals and propose a genetic mechanism to

explain the unknown etiology of RPL. With the completion of human genome project

it is imperative to understand the genetic basis of diseases and to identify the

population and race polymorphism. Since pregnancy is a complex process and in

about 50% of the RPL cases the cause is unidentified, it is essential to explore the

contribution of the genetic variations in RPL.

Introduction



1.1 Recurrence risk for RPL

About 10–15% of all clinically recognized pregnancies end in a miscarriage globally.

It has been reported that the recurrence risk increases with each successive pregnancy

loss. Risk of pregnancy loss in women with atleast one live born infant without any

prior fetal loss is 12 % for the next pregnancy; whereas the percentage of recurrence

risk for those with at least one, two and three prior fetal loss is 24, 26 and 32%

respectively. However for women with no live born infant and with 2 or more fetal

losses the recurrence risk for the next pregnancy is 40 -45 % (8). The recurrence risk

rates for pregnancy loss are indicated in Fig 1.1

01 2 3 2*

0

5

10

15

20

25

30

35

40

Recurr

ence ris

k (%

)

Recurrence risk in RPL

* - With no live children (Source data – Arjun et al 2000)(8)

Number of fetal Loss

Fig 1.1: Recurrence risk rates for RPL

Introduction



1.2 Indian Scenario

A reliable estimate on the rate of pregnancy loss in the Indian context is deficient, as

the registration of marriages, births and deaths are usually incomplete. However an

indirect source that provides information on the incidence of pregnancy loss is the

multicentric study on genetic counseling carried out by the task force of Human

Genetics under the auspices of the Indian Council of Medical Research (ICMR) from

1992 – 1995 (9). The data of ICMR report on the percentage of different disorders

referred for genetic counseling is shown in Fig 1.2. From the data it is evident that

RPL is the topmost disorder referred for genetic counseling in India. Hence, it is

essential to arrive at a lucid understanding of the causes of RPL in the Indian context,

which are hitherto unexplored.

0

2

4

6

8

10

12

14

Repeate

d

abort

ions

Identifiable

Syndro

mes

Chro

mosom

al

dis

ord

ers

Menta

l

Reta

rdation

Inte

rsex

Hig

h r

isk

pre

gnancy

Hem

ato

logic

al

dis

ord

ers

Neura

l tibe

defe

cts

Congenital

anom

alies

Meta

bolic

dis

ord

ers

Pri

mary

mic

rocephaly

Skele

tal

Dyspla

sia

Perc

enta

ge

Fig 1.2: Categories of patients referred for

genetic counseling in India

ICMR report

1995(9)

Introduction



1.3 Etiological factors for RPL

The established etiological factors that might result in RPL fall into two broad

categories namely embryonal defects like chromosomal abnormality which inhibit the

embryo from implanting or developing and alterations in maternal environment in

which the fetus grows. This includes infection, anatomic, endocrine, genetic and

immunological abnormalities. Fig 1.3 represents in a nutshell these two categories of

etiological factors.

1.3.1 Embryonal defects

Implantation usually occurs after 8-10 days of fertilisation in most healthy

pregnancies. Delay in implantation enhances the proportion of early embryo loss. It

has been shown that chromosomal abnormalities in the conceptus are characteristic of

spontaneous abortions. Chromosomal abnormalities were responsible in about 50% of

first trimester miscarriages, 5% of late pregnancy losses and 0.5% of live births.

Cytogenetically abnormal embryos are usually aneuploid because of sporadic events,

such as meiotic non-disjunction, or polyploid from fertilization abnormalities (11).

Embryonal

defects

Chromosomal

abnormalities

Alterations in

maternal

environment

1) Anatomical 2) Endocrine 3) Infection 4) Genetics 5) Immunology

Recurrent Pregnancy Loss

Fig 1.3: Etiological factors for recurrent pregnancy loss

Meka et al 2006 (10)

Introduction



Table 1.1 summarizes the different types of chromosomal abnormalities of the

conceptus associated with recurrent pregnancy loss.

1.3.2 Alterations in the maternal Environment

The maternal environment alterations include anatomic uterine defects, endocrine

anomalies, hormonal imbalances, infection, metabolic dysfunction nutritional

deficiencies, immune disorders and genetic factors.

1.3.2.1 Anatomic uterine defects

Anatomic uterine defects are known to cause obstetric complications including RPL,

preterm labor and delivery. The common anatomic defects associated with RPL are

mullerian duct fusion in the uterus, double uterus, uterine septum, endometrial polyps

and sub-mucous fibroids. Impaired vascularization and fetal growth restriction due to

uterine distortion are common reasons for pregnancy loss. Although congenital

uterine abnormalities have been associated most often with second trimester

pregnancy loss, 15% of women with recurrent early pregnancy loss also exhibit these

abnormalities (12).

1.3.2.2 Hormonal and metabolic disorders

Hormonal imbalances are believed to play a role in at least 10-20% of recurrent

pregnancy losses. Ovulation, implantation and the early stages of pregnancy depend

on an integral maternal endocrine regulatory system. Deficient secretion of

progesterone or a poor endometrial response to progesterone results in Luteal Phase

Deficiency (LPD) which leads to abnormal endometrial development, thus affecting

implantation and thereby enhancing the risk of pregnancy loss. In addition, thyroid-

gland dysfunction, type I diabetes mellitus and poly cystic ovary syndrome have also

been associated with the risk of RPL (13).

0

5

10

15

20

25

30

Percentage

G*01011 G*01012 G*01013 G*01014 G*0103 G*0104 G*0105N

HLA G Alleles

Controls

Cases

Fig 3.5: Distribution of HLA G polymorphism in RPL

and control women

Controls (N = 80) Cases (N =104)

HLA G alleles Number Allele frequency Number Allele frequency

G*01011 22 0.14 22 0.11

G*01012 24 0.15 30 0.14

G*01013 23 0.14 29 0.14

G*01014 16 0.10 17 0.08

G*0103 42 0.26 45 0.22

G*0104 27 0.17 34 0.16

G*0105N 6 0.04 31 0.15

P< 0.001

Table 3.4: Frequency distribution of the various HLA G alleles in

RPL and control women

Atleast one allele Controls Cases

G *01011 0 2

G *0103 7 7

G *0104 1 11

P< 0.03

Table 3.7: Association of HLA G alleles that alter the

amino acid sequence and RPL

0

10

20

30

40

50

60

70

80

90

Percentage

HLAG*01011 HLAG*0103 HLAG*0104

HLA G alleles

Controls

Cases

Fig 3.6: Distribution of HLA G alleles that alter

the amino acid sequence

0

5

10

15

20

25

30

35

40

45

50Genotype frequency

I/I I/D D/D

HLAG genotypes

Controls

Cases

Fig 3.7: HLA G I/D polymorphism in RPL and control women

Genotype Genotype frequency OR 95% CI P

Controls

(N=80)

Cases

(N=104)

I/I 27 23 1.0

I/D 28 49 2.34 1.13 - 4.88 <0.05

D/D 25 32 1.65 0.76 to 3.57 NS

NS – Non significant

Table 3.9: Association of HLA G I/D polymorphism and RPL

0

10

20

30

40

50

60

70

80

Percentage

Control Cases

Antipaternal cytotoxic antibodies and RPL

Present

Absent

Fig 3.9 Antipaternal cytotoxic antibodies and RPL

APCA Controls

(N = 30)

Cases

(N = 30) P

Present 22 7

<0.001

Absent 8 23

Table 3.13: Distribution of APCA in RPL

and control women

0 20 40 60 80

Percentage

0

atleast 1

0

atleast 1

Present

Absent

Distribution of HLA G allele sharing and APCA in RPL women and

controls

Cases

Controls

Fig 3.10: Distribution of HLA G allele sharing and APCA in RPL

and control women

APCA HLA sharing Controls

(N=30)

Cases

(N=30)

P

Present 0 22 6

NS At least 1 allele 2 1

Absent 0 5 6 0.04

At least 1 allele 1 17


Table 3.14: Association of APCA and HLA allele sharing in RPL

Introduction



* Arjun et al 1996(8)

Type Chromosomal

constitution

Percentage*

Normal 46 XY or 46 XX 54.1

Triploidy

69, XXX 2.7

69, XYX 0.2

69, XXY 4.0

Other 0.8

Total 7.7

Tetraploidy

92, XXXX 1.5

92, XXYY 0.55

Others 0.55

Total 2.6

Monosomy X 8.6

Structural abnormalities 1.5

Sex chromosomal

polysomy

47, XXX 0.05

47, XXY 0.15

Total 0.2

Autosomal monosomy 0.1

Autosomal trisomy 22.3

Double trisomy 0.7

Mosaic trisomy 1.3

Other abnormalities 0.9

Table 1.1 - Chromosomal abnormalities in the aborted embryo and fetuses

Introduction


loss in South Indian Population

7

1.3.2.3 Infections

Infected or inflamed uterine lining would be a hostile environment for implantation

and development of the embryo. Microbial infection of the uterus or vagina with

toxoplasma, listeria, cytomegalovirus and parvovirus are known to cause sporadic

pregnancy loss, but no infectious agent has been proven to cause RPL. In addition,

chronic herpes simplex viral infection is a possible cause of RPL. Fetal infection may

lead to either fetal death or malformations incompatible with viability (14).

1.3.2.4 Nutritional factors

The deficiency of vitamin A is a cause of increased infant mortality. Low

concentrations of dietary and circulating folate are associated with increased risks of

preterm delivery, reduced infant birth weight and fetal growth retardation. Folate

deficiency results in elevation of blood homocysteine and increased homocysteine

concentrations have been associated both with spontaneous abortion and pregnancy

complications (15).

1.3.2.5 Immunological causes

The immune factors associated with pregnancy loss are classified as autoimmune and

alloimmune factors. The autoimmune factors include anti-phospholipid and anti-

nuclear antibodies. Phospholipids are fatty molecules on cell membranes that help to

maintain the proper balance between bleeding and clotting. Antiphospholipid

antibodies block the phospholipids from regulating blood flow and clotting, resulting

in the formation of blood clots in the placenta leading to pregnancy loss (16).

Antinuclear antibodies are autoantibodies to the DNA which lead to inflammation in

the placenta. Alloimmune response refers to the maternal immune response to

antigens of placental or fetal tissues. In normal pregnancy, placenta and the growing

embryo are not entirely “self” but rather is a result of both the maternal and paternal

genetic heritages, referred to as a semi-allograft. Though the exact mechanism that

allows the embryo to escape immune rejection is unknown, the human leukocyte

antigen sharing between partners, decrease in natural suppresser cells in the uterus,

serum blocking factors that protect the placenta from paternal antigens and

Introduction


loss in South Indian Population

8

antileukocytotoxic antibodies against paternal leukocytes have been proposed for

successful pregnancy. Alterations in any of these immune mechanisms could result in

RPL (17).

1.3.2.6 Genetic factors

Genetic factors account for approximately 5% of pregnancy loss (18). Cytogenetic

analyses in blood lymphocytes of couples with RPL in the Indian population have

revealed an overall 4-5% chromosomal abnormality (19, 20). Certain chromosomal

abnormalities seen in couples, which are considered to be a significant cause of RPL

is summarized in Table 1.2.

X chromosome inactivation (XCI), an essential random phenomenon

for dosage compensation in females occurs such that maternally and paternally

derived X chromosomes are inactivated with approximately the same frequency.

Skewed X chromosome inactivation is a condition where there is preferential

inactivation of either the maternal or the paternal derived X chromosome. An

extremely skewed XCI of ≥ 90 % has been reported in females with a history of RPL

(21, 22). Because there are many potential causes of skewed XCI, the reason for an

association between RPL and skewed XCI is not clear and may be heterogeneous. X

linked mutations have been clearly associated with skewed XCI and RPL in some

families (23).

1.3.2.7 Other risk factors

Advanced maternal age is a significant risk factor for pregnancy loss. Fetal loss is

high in women in late thirty years or older, irrespective of reproductive history. The

risk of ectopic pregnancy and stillbirth also increase with increasing maternal age

(24).

Smoking and alcohol are also cited as risk factors for RPL. Organohalide pesticides

and organic solvents have been implicated in RPL. Sporadic miscarriages have also

0

10

20

30

40

50

60

70

80

90

100

H+/W-

(inh)

W+/H-

(inh)

H+/W-

(act)

W+/H-

(act)

Controls

Cases

Fig 3.12 Sharing of activating and inhibitory KIR genes

in RPL and control couples

Category Controls (N=80) Cases (N=104) P

H+/W- (inh) 20 51 <0.001

W+/H- (inh) 62 44 <0.001

H+/W- (act) 42 45 NS

W+H- (act) 77 91 NS


Table 3.22: Association of activating and inhibitory KIR gene

sharing in RPL

H+/W- : Wife lacks the inhibitory receptor possessed by the husband

W+/H- : Husband lacks the inhibitory receptor possessed by the wife

H+/W- : Wife lacks the activating receptor possessed by the husband

W+/H- : Husband lacks the activating receptor possessed by the wife

Introduction



* Dubey et al 2005 (20)

Type of abnormality Chromosomal Constitution* Sex

Structural chromosomal abnormalities – (71%)

Reciprocal

Translocation

46,XX t(1;5) (p33:q35) F

46,XX t(2;11) (q21:q24) F

46,XX t(3;11) (q26:11pter) F

46,XX t(3;17) (p22:p13) F

46,XX t(6;X) (q32:q22) F

46,XX t(6;12;13)

(q22:q21:q23:q12)

F

46,XX t(6;18) (q27:q21) F

46,XY t(7;8) (q11:p11) M

46,XY t(7;13) (pter:q13) M

46,XX t(8;15) (q12:p11) F

Robertsonian

translocation

46,XX t(13;14) F

46,XX t(21;22) F

46,XX t(22;22) F

Deletion + marker 46,XYdel(3) (pter → q25) + marker M

46,XYdel(10) (pter → q 22) + marker M

Inversion 46,XY inv (4) (p15:q13) M

Numerical chromosomal abnormalities – 29%

47,XXX / 46,XX F

47,XXY / 46,XY M

47,XXX / 46,XX / 45,XO F

47,XXX / 46,XX F

46,XX / 45,XO F

Other Polymorphic Variants

Polymorphic

chromosomal variants

Yq+, Yq-, Pericentric 1qh+, inv (9), 9qh+, 15p+, 16qh+,

22p+, Fragile sites

Table 1.2 - Chromosomal abnormalities in blood lymphocytes

of couples with RPL

Introduction



been reported with intrauterine diethylstilbestrol exposure. Though psychological

factors, stress and grief have been implicated in RPL their exact role is unclear. (25)

Though all the above described etiological factors have been proposed to attribute to

the risk of RPL, the unexplained etiology in 50% of the cases stressed the need to

identify the role of additional genetic alterations which might collectively result in

RPL.

1.4 Genetic polymorphisms in RPL

The search for genetic markers of disease susceptibility often utilizes the candidate

gene approach, where a gene is targeted based on the properties and metabolic

pathways of its protein product (26). When these genes are polymorphic and the

variants are distributed differently across populations, interest in them increases since

variation in the DNA sequence could alter protein function and result in variations in

disease risk. Recent studies have revealed an association between RPL and genetic

polymorphisms in metabolic enzymes, cytokines, coagulation factors,

methylenetetrahydrofolate reductase and histocompatibility antigens.

1.4.1 Genetic polymorphisms in Immunomodulatory genes

Human immune system is well known for its ability to discriminate between non-self

antigens like that of infections and solid tissue transplantation over the self antigens.

Over the years it has become evident that highly polymorphic molecules encoded by

genes located on chromosome 6 termed Major Histocompatibility Complex (MHC),

referred to as Human Leukocyte Antigens (HLA) in humans, play a key role in

immune rejection (27, 28). According to the classic transplantation rules, MHC

mismatched transplants are rejected by the recipient, whereas MHC matched

transplants are accepted. A notable, but as yet unresolved exception from the classic

transplantation paradigms is pregnancy, where a semiallogeneic fetus thrives in the

mother’s womb. Though a detailed mechanism behind the maternal–fetal

immunotolerance remains elusive, specific and direct interactions between maternal

and fetal cells is suggested to play an important role (29). Recent evidence strongly

Introduction



supports active immune tolerance of the fetus during pregnancy (30, 31). The major

genes regulating this immunomodulation include HLA, Natural Killer cell receptors

and cytokines.

1.4.1.1.1 Human Leukocyte Antigen

HLA genes are the main genetic determinants of the repertoire of individual immune

responses. HLA complex plays a critical role throughout pregnancy by influencing

gamete development, embryo cleavage, blastocyst and trophoblast formation,

implantation, fetal development and survival. The HLA complex contains

approximately 4 Mb of DNA on chromosome 6p21 and is composed of three classes

namely class I, II and III (32, 33) (Fig 1.4). The expressed class I genes are

subdivided into class Ia, which includes HLA-A, -B, and -C, and class Ib, which

includes HLA-E, -F, and -G. HLA class II includes the DR, DQ and DP (34, 35).

Human trophoblast cells express one class Ia molecule (HLA-C) and all three class Ib

molecules. Of the HLA class Ib molecules expressed by trophoblast cells, HLA-G

was the first identified antigen of great interest and currently a focus of experimental

evaluation.

A C DR B E G F DQ DP

Class II Class III Class I

Fig 1.4 HLA Complex

(Darkly stained boxes indicate less polymorphic genes)

Introduction



1.4.1.1.2 Structural features of HLA-G

Although the genomic structure of HLA-G is similar to other class I genes, it is unique

in most respects. HLA-G gene consists of eight exons encoding a signal peptide (exon

1), the α1, α2 and α3 domains (exons 2, 3, and 4 respectively), the transmembrane

domain (exon 5) and the intracellular domains (exons 6 and 7) (Fig. 1.5). A premature

stop codon in exon 6 results in a truncated cytoplasmic segment of the HLA-G

protein, which results in the loss of 19 amino acid residues which are highly

conserved in other classical HLA-loci (36). The functional significance of the

shortened cytoplasmic tail of HLA-G is unknown.

Another unique feature of HLA-G is that it encodes multiple isoforms as a result of

alternative splicing. Seven alternatively spliced transcripts have been identified, of

which four are predicted to encode membrane bound proteins (HLA G1, HLA G2,

HLA G3, HLA G4) and three are predicted to encode soluble proteins (HLA G5,

HLA G6, HLA G7). Five of the seven transcripts that result from alternative splicing

are shown in Fig.1.5. The full-length isoform HLA-G1 is structurally similar to the

other class I genes, except for the truncated cytoplasmic tail. The G2 isoform results

from the removal of exon 3 and subsequent homodimerization to form a HLA class II-

like structure (34, 37). These two isoforms with an inclusion of intron 4 sequences in

the mature mRNA with an additional of 21 amino acids are expressed as soluble

secreted proteins known as HLA G5 and G6, respectively (38). HLA G3 results from

the removal of exons 3 and 4. HLA G4 and G7 mRNAs are not abundant in placentas.

Exon 4 (encoding the α 3 domain) is spliced out of the HLA G4 transcript; the HLA

G7 transcript includes exon 2 and part of intron 2 and is predicted to encode a small

soluble isoform. The expression of class Ia antigens is ubiquitous whereas expression

of class Ib antigens may be tissue/ organ-specific and/or conditional (39, 40).

1.4.1.1.3 HLA G Polymorphism

HLA G has relatively little polymorphism in its coding region compared to the

classical class I genes. 13 polymorphisms have been identified in exons 1–4 and one

in the 3’UTR (Fig. 1.6). Polymorphisms at codon 31 in the α 1 domain (Thr3Ser),

13

αααα2 – domain


Cytoplasmic

domain

3” UTR

Exon

1

2

3

5

6

7

8

Signal peptide


Trans

membrane

region

e1 2 3 4 5 6 8

e1 2 4 5 6 8

e1 2 5 6 8

e1 2 3 5 6 8

e1 2 4 4 5 6 i

e1 i

e1 2 3 4 5 6 8 i

5 6 8

α3

α2 α 1

α3

α2 α 1

α3

α 1

α 1

α 2

α 1

α 1

α3

α 1

5’

3’

3’

5’

3’ 5’

Full length

HLA G gene HLA G mRNA HLA G Protein

5’ 3’

5’ 3’

3’ 5’

5’ 3’

Exon 3 spliced out

Exon 3 & 4 spliced out

Exon 4 spliced out

Retains intron 4 –

Stop codon

Retains intron 4 –stop codon

& exon 3 spliced out

Retains intron

2 – stop codon

HLA – G1

HLA – G2

HLA – G3

HLA – G4

sHLA – G5

sHLA – G6

sHLA – G7

Fig 1.5: HLA-G gene structure, spliced transcripts and

isoforms (Source data - Hviid et al 2003) (50)

Introduction

A study on genetic polymorphisms associated with unexplained recurrent pregnancy loss in

South Indian Population

14

at codon 110 in the α 2 domain (Leu3Ile), and at codon 258 in the α 3 domain

(Thr3Met) result in an amino acid substitution. Polymorphisms at nucleotide 15 and

36 in exon 1, at codons 35, 57 and 69 in exon 2, at codons 93, 100 and 107 in exon 3,

and at codon 188 in exon 4 do not alter the amino acid sequence of the protein. In

contrast to the class Ia HLA loci, amino acid substitutions at codons 31 and 110 in the

α 1 and α 2 domains, are conservative changes that occur in residues that are not

predicted to interact with bound peptide or T cell receptor (41). The third amino acid

polymorphism at codon 258 is a non-conservative substitution in the α 3 domain that

is highly conserved in the class Ia genes (39). It is located in the pleated sheet

structure of the α 3 domain, where it might affect recognition of CD8 in the HLA G1

and G5 isoforms, or binding to CD4 in the HLA G2 and G6 isoforms (42). A single

base pair (bp) deletion at nucleotide 1597 causes a frameshift at amino acid 130 (43),

resulting in nonfunctional HLA G1 and G5 proteins (41, 44). This null allele has been

associated with increased risk for recurrent pregnancy loss (45, 46) suggesting that

HLA G1 and/or G5 proteins play an important role in the maintenance of pregnancy

and that reduced levels of one or both is a risk factor for RPL.

The polymorphisms that alter the protein sequence define five alleles, called G*0101,

G*0103, G*0104, G*0105N, and G*0106. Silent variation within these allelic classes

defines subtypes, referred to as G*01011, G*01012, etc (Table 1.3). A 14 bp

insertion/deletion polymorphism in the untranslated exon 8 of the HLA G gene was

first described by Harrison and colleagues (47), but has recently been shown to

influence mRNA transcript size and stability. The presence of the 14 bp insertion

allele generates a 92 bp deletion in the 3’UTR of the mRNAs, possibly because it acts

as a cryptic splice site (48). Transcripts with the 92 bp deletion were associated with

more stable mRNA in JEG-3 cells and in an M8 cell line (49). Further, the relative

abundance of the alternatively spliced transcripts may be influenced by

polymorphisms in HLA G (50).

Introduction

A study on genetic polymorphisms associated with unexplained recurrent pregnancy loss in South Indian Population

15

** - Polymorphism at codon 188 of Exon 4

Allele Exon 2 Exon 3

Codon 31 Codon 54 Codon 57 Codon 69 Codon 93 Codon 107 Codon 110 Codon 130

G*01011 ACG CAG CCG GCC CAC GGA CTC CTG

G*01012 ACG CAG CCA GCC CAT GGA CTC CTG

G*01013 ACG CAG CCA GCC CAC GGT CTC CTG

G*01014 ACG CAG CCG GCT CAC GGA CTC CTG

G*01015 ACG CAG CCG GCC CAC GGT CTC CTG

G*01016** ACG CAG CCG GCC CAC GGA CTC CTG

G*01017 ACG CAG CCA GCC CAT GGT CTC CTG

G*01018 ACG CAG CCA GCC CAC GGA CTC CTG

G*0102 ACG CGG CCG GCC CAC GGA CTC CTG

G*0103 TCG CAG CCG GCC CAC GGA CTC CTG

G*01041 ACG CAG CCA GCC CAC GGA ATC CTG

G*01043 ACG CAG CCG GCC CAC GGA ATC CTG

G*0105N ACG CAG CCA GCC CAT GGA CTC -TG

Table 1.3: HLA G Polymorphism

Introduction



1.4.1.2.1 Natural Killer cells

Natural killer (NK) cells represent a subset of lymphocytes found in a variety of

tissues in humans, including deciduas (51). Because of their increased presence and

direct contact with trophoblast, NK cells have been considered to be a crucial cell

population for pregnancy (52). The property of NK cells to recognize and lyse target

cells and discriminate them from normal cells is mediated by the activating and

inhibiting NK receptors (53). The NK cell receptors (NKRs) belong to three main

families: the killer immunoglobulin-like receptors family (KIR) (54, 55), the C-type

lectin family (CD94 ⁄ NKGs) and the immunoglobulin-like transcripts (ILTs or LIRs)

(56, 57). A number of NKRs of each family as well as their ligands have been

identified and it is known that the functions of NK cells are repressed by specific

recognition of HLA class I molecules. Multiple combinations of NKRs are co-

expressed by individual NK cell clones. The NKR repertoire varies among different

individuals and human NK cells employ receptors of all receptor families for the

recognition of different human leukocyte antigen (HLA) class I molecules. Further,

every mature NK cell is predicted to bear at least one type of dominant inhibitory

receptor for a self-MHC class I product, to prevent auto-reactivity against normal

cells (58, 59, 60).

1.4.1.2.2 Killer cell immunoglobulin-like receptors (KIR)

In humans, an important superfamily of NK receptor is KIR (Killer cell Ig-like

receptor). The KIR are members of the immunoglobulin (Ig) super gene family

currently comprised of 15 genes and two pseudogenes encoded within a 100-200 Kb

region of the Leukocyte Receptor Complex (LRC) on human chromosome 19 (Fig

1.6) (61, 62). Based on the number of Ig-domains in the extracellular region, KIR

receptors can be divided into either three Ig domains containing KIR (KIR3D) or two

Ig domains containing KIR (KIR2D) (63, 64). Depending on the cytoplasmic tail

length, they can be further grouped into either long (L) tail KIRs having inhibitory

function, or short (S) tail KIRs with activating function. Long tail KIRs have one or

two immunoreceptor tyrosine-based inhibitory motifs (ITIM) and transducer

inhibitory signals. In contrast, the short tail KIRs have no ITIM motif but possess a

charged residue in the transmembrane region that mediates association with DAP12

Introduction



(DNAX-activating protein of 12kDa). The DAP12 contains immunoreceptor tyrosine-

based activation motif (ITAM) and transduces activating signals (59).

1.4.1.2.3 KIR Polymorphism

A peculiarity of KIR genetics is their haplotypic polymorphism, i.e. the presence or

absence of some KIR genes on individual chromosomes (57). There is an extensive

polymorphism among KIR haplotypes, which differ not only in nucleotide

sequence but also in gene content. Different haplotypes carry different numbers of

KIR genes, some with few or no activating KIR’s (A haplotypes) and others with

several activating KIR’s (B haplotypes). In addition, each NK cell expresses its

own repertoire of KIR genes.

1.4.1.2.4 KIR Haplotypes

All known KIR haplotypes are flanked at their centromere by KIR3DL3 and at their

telomeric end by KIR3DL2, together with the centric KIR3DP1 and KIR2DL4. These

constitute the framework genes, which limit two regions of variable KIR gene content

where the remaining KIR genes are located (61, 62, 65). All KIR genes are arranged

in a head to tail fashion approximately 2.4 Kb apart from each other (62). Based on

their gene content two kinds of KIR haplotypes, A and B have been described (Fig

1.7). The “A-group haplotypes” are relatively simple, consisting of 7 functional

genes. They include inhibitory KIR genes for all major HLA class I specificities, and

have one or two activating KIRs (KIR2DS2 and/or KIR2DS4). The “B-group

haplotypes” contain 9-12 genes. They are marked by KIR2DL5 gene (66) and most of

the KIR genes in the B-group haplotypes are activating type. B-group haplotypes may

not carry inhibitory KIR genes for some HLA specificities. Both groups of haplotypes

conserve only three ‘framework’ genes (62, 67).

Introduction



During pregnancy, maternal–fetal interface is characterized by an unusual

combination of HLA and NKR expression on extravillous cytotrophoblasts (EVT) and

the pattern of maternal leukocyte distribution in the decidua. Further, it is reported

that the interaction between the HLA G and the KIR play an important role in the

maintenance of pregnancy.

1.4.1.3 Cytokine gene polymorphisms

1.4.1.3.1. Interleukin 1 (IL – 1 Receptor Antagonist)

Interleukin 1 (IL-1) is a central proinflammatory cytokine, produced by monocytes,

macrophages, and epithelial cells (68). Secretion of IL-1 leads to a proinflammatory

cascade, including TH-1 proliferation, production of tumor necrosis factor (TNF)-α,

interferon (IFN)-γ, IL-2, and IL-12. The level of IL-1 is modulated by an endogenous

factor, IL-1 receptor antagonist (RA). An 86–base pair tandem repeat in intron 2 of

IL-1 RA gene has been associated with increased risk of RPL (69).

3D

L3

3D

P1

2D

L4

3D

L2 2D

S4 3D

L1

2D

L3

2D

P1

2D

L1

Group A

3D

L3

3D

P1

2D

L4

3D

L2 2D

S2

3D

S1 2D

L2

2D

S5

2D

S3

2D

S1

2D

L5

Group B

Fig-1.7: Genomic organization of two groups of KIR haplotypes

Genes conserved in both groups of haplotypes are shown in dark boxes.

Introduction



1.4.1.3.2. Transforming Growth Factor Beta 1 (TGF Beta 1)

TGF-beta1 is an immunosuppressive cytokine. This cytokine may contribute to

regulation of maternal immune responses directed against the fetal allograft, and

thereby prevent immunological rejection of the fetus. Studies have shown that TGF-

beta1 negatively regulates the primary immune response induced by Th1 but not Th2

memory cells (70, 71). Several polymorphisms have been reported in TGF beta1 and

certain polymorphic alleles are suggested to be associated with an elevated serum

level of TGF beta1 both in vitro and in vivo (72, 73). Polymorphisms at nucleotides

29 and 74 resulting in T to C and G to C substitutions respectively are reported to

affect TGF beta1 secretion and have been implicated in RPL (74)

1.4.1.3.3. Tumor necrosis factor alpha (TNF alpha)

Tumor necrosis factor is a potent cytokine with a wide range of proinflammatory

activities. It is classically produced by monocytes/macrophages although other cell

types, such as T and B cells, also produce significant amounts (75, 76). Circulating

levels of TNF alpha are higher in patients with pregnancy loss compared to those with

a successful pregnancy, suggesting that this cytokine may be an etiologic factor in

RPL (77, 78, 79). Additionally, functional polymorphisms at position -308 and -863

in the promoter region of the human TNF alpha gene have been reported to be

associated with altered TNF alpha activity (80). Though a few studies have reported

these polymorphisms to increase the risk of RPL, others have found no association

(81, 82)

The various candidate genes of the immune system, their function and the effect of

their polymorphisms in RPL have been tabulated (Table 1.4)

1.4.2 Genetic polymorphisms in placental networking and vascular

remodeling

Successful pregnancy requires the development of a complex maternal and fetal

vascular network to support increasing oxygen and metabolic demands of the growing

fetus. There are three stages in human placental vascular development: an initial stage

Introduction


20

Candidate Genes Gene loci Polymorphism Gene function Effect of Polymorphism

HLA-G

(Aldrich et al 2000) (45)

6p21 HLA G*010103

HLA G*0105N

Immunomodulation at the

feto maternal interface

Low secretor allele

HLA –DR

(Takakuwa et al 2003)(83)

6p21 HLA DRB1*15

HLA DRB1*04

Immune response to the

foreign antigens

Maternal immune response

against the fetus

HLA –B

(Christiansen et al 1997)(84)

6p21 Bw4 Influence both T-cell-

mediated and NK-cell-

mediated rejection

Maternal immune response

against the fetus

NKRs

(Clark et al 2005) (52)

19q13 2DL1, 2DL2, 2DL3 Protect trophoblast from

maternal NK cells attack

and to control trophoblast

migration and placentation.

NK cell mediated cytotoxic

effect on trophobalst cells

TNF – alpha

(Ragupathy et al 2000) (78)

6p21 - 863 C/A

- 308 G/ A

Potent cytokine with a

wide range of

proinflammatory activities.

Associated with altered TNF

promoter activity

Table 1.4: Polymorphisms in immunomodulatory genes reported in RPL

Introduction


21

TGF Beta –1

(Amani et al 2004)(74)

19q13

-800 G/A

-509 C/T

Modulation of cellular

growth and differentiation,

immunoregulation and

extra cellular matrix

formation

Altered production of TGF

beta

Il-1 RA (Tarlow

et al 1993) (69)

2q14 86bp Tandem

repeat in Intron 2

Competitive antagonist for

IL-1

Altered Function

IL-18

(Dinarello et al 1998)(68)

11q22 -607 (C/A)

-137 (G/C)

Immune regulation at

maternal-fetal interface

Implantation failure in cases

of absence or over activation

Introduction



of vasculogenesis, subsequent branching and then non branching angiogenesis (85,

86). Vasculogenesis involves the de novo formation of new vessels, whereas

angiogenesis refers to the formation of capillaries from preexisting vessels (87). The

development of a normal functioning placental vascular network requires a

remarkable degree of coordination between different cell-specific growth factors and

signals exchanged between these cells (88). Pregnancy also requires a balance

between coagulation and fibrinolysis in order to avoid excess fibrin accumulation in

placental vessels and intervillous spaces as well as to secure fibrin polymerization and

stabilization of the placental basal plate. Adequate fine-tuning of fibrinolysis is

mandatory to prevent hemorrhage (89, 90). Minimal alterations in the fibrinolysis

cascade leading to either hypo or hyperfibrinolysis are also suspected to interfere with

placentation in early pregnancy (91).

Although numerous factors have been implicated in placental vascular network,

recent observations including gene knockout studies in mice have led to the

identification of the major factors regulating the placental vascular network. It

includes the growth factors like Vascular Endothelial Growth Factor (VEGF), which

helps in the cell proliferation and cell differentiation (92), Angiotensin Converting

Enzyme (ACE), which is involved in the physiological remodeling of spiral arteries

(90) and Methylene Tetrahydrofolate Reductase (MTHFR), the methyl donors for

vascular development (93) (Fig 1.8). The other important genes in this pathway

include Factor V Leiden, PAI1 and prothrombin.

1.4.2.1.1 Vascular endothelial growth factor (VEGF)

VEGF is an angiogenic factor and a prime regulator of endothelial cell proliferation. It

exhibits a crucial role in physiological vasculogenesis and vascular permeability (94).

It belongs to Platelet derived growth factor (PDGF) super family and has more than

10 isoforms namely, VEGF-121, VEGF-165, VEGF-189, VEGF-206, -B, -C, -D, -E,

PIGF-I and PIGF-II, ranging in weight from 35-44 kDa. Each binds to a specific

combination of endothelial cell surface receptor known as VEGFR-1, -2 and -3 (95).

Circulating VEGF binds to VEGFR on endothelial cells, triggering a tyrosine kinase

pathway leading to angiogenesis (96). VEGF also protects endothelial cells from

Introduction



apoptotic cell death during embryonal development and withdrawal of VEGF by

targeted inactivation of the VEGF gene is found to result in massive endothelial cell

apoptosis, leading to severe hemorrhage (97). Thus VEGF/VEGFR system is one of

the best characterized regulators of angiogenesis (98).

1.4.2.1.2 Polymorphisms in VEGF gene

VEGF gene is located on chromosome 6p21 and is comprised of a 14 kb coding

region with 8 exons and 7 introns (Fig 1.9). Many polymorphisms have been

identified in the VEGF gene and a few of them have been correlated with variation in

VEGF production (99,100).

1.4.2.2.1 Angiotensin converting enzyme (ACE)

ACE is a zinc-metalloproteinase occurring both as a membrane-bound ectoenzyme on

the surface of vascular endothelial and renal epithelial cells as well as a

circulating

enzyme in the plasma (101,102). ACE is a key component of the Renin Angiotensin

system (RAS), which regulates blood pressure, fluid and electrolyte balance (103). In

addition, it plays an important role in activating angiotensin I to angiotensin II.

Evidence from animal models have suggested that angiotensin II stimulates

neovascularization (104,105) by promoting arteriolar smooth muscle cell proliferation

(106). Further, angiotensin II may act as a mitotic factor by inducing or regulating

gene expression in cell cycle progression (107)

Placental and fetal membranes are important sites for the synthesis of the RAS

components. In vivo and in vitro studies suggest the role of RAS in haemostasis

through different mechanisms, including an influence on fibrinolysis, platelet

aggregation and blood clotting activation (108, 109). Studies have reported an

association between the ACE genotypes and increased risk of thrombophilia (110), a

condition predisposing to adverse pregnancy outcomes. Moreover, ACE by

bradykinin degradation reduces nitric oxide levels, thereby contributing to endothelial

dysfunction. In addition to regulating the vasomotor functions, RAS is also involved

Introduction



in key events of the inflammatory process (111) by increasing vascular permeability

and contributing to the recruitment of inflammatory cells.

1.4.2.2.2 Polymorphisms in ACE gene

ACE gene located on 17q23, spans 21 kb and consists of 26 exons and 25 introns (Fig

1.10). About 47% of interindividual variability of plasma ACE concentration is

determined by the presence [insertion (I)] or absence [deletion (D)] of a 287-bp

Alu-

type sequence in intron 16 of the ACE gene (112). Homozygous I allele displays as

low as half of the ACE level compared with the homozygous

D allele, whereas the I/D

heterozygotes display an intermediate level (107). Though the exact mechanism of

ACE polymorphism on the serum ACE level has not been identified, studies have

suggested that the insertion/deletion may be in linkage disequilibrium with regulatory

elements of the ACE gene, or that the insertion itself might modify the splicing

process of the ACE precursor mRNA by interfering with the lariat formation (113).

1.4.2.3.1 Methylenetetrahydrofolate reductase (MTHFR)

MTHFR catalyzes the reduction of 5, 10-methylenetetrahydrofolate to 5-

methyltetrahydrofolate, the predominant circulatory form of folate and the carbon

donor for remethylation of homocysteine to methionine (114,115). Methionine is

involved in the formation of S-adenosylmethionine, a principal methyl donor in cells

for DNA methylation. S-adenosyl methionine is also an allosteric inhibitor of MTHFR

and an activator of cystathionine ß-synthase. Although the effects of folate

metabolism are still incompletely understood, there is growing evidence that normal

MTHFR activity is essential to maintain the pool of available circulating folate and

methionine as well as prevent accumulation of homocysteine (116).

Hyperhomocysteinemia and reduction in S-adenosylmethionine that might result from

altered MTHFR activity are associated with the impaired DNA methylation and gene

expression leading to defective chorionic villous vascularization.

1.4.2.3.2 Polymorphisms in MTHFR gene

The MTHFR gene is mapped to chromosome 1p36 (Fig 1.11). The cDNA sequence is

2.2 kilobases long and consists of 11 exons. A common functional polymorphism

Introduction



which alters the MTHFR activity is the C to T substitution in the 677 nucleotide

(117,118). Which results in alanine to valine substitution within the catalytic domain

of MTHFR which results in decreased enzyme activity.

1.4.2.4 Activated protein C and Factor V Leiden

Protein C is a key component in the anticoagulant pathway which when activated

inhibits the actions of coagulation factors V and VIII. Congenital

APC resistance is

almost exclusively due to a single point mutation (G A) at nucleotide position 1691

in the factor V gene, which results in a mutated form of factor V, known as factor V

Leiden (119). Mutated factor V is resistant to inactivation by APC, resulting in

increased thrombin generation and a hypercoagulable state leads to increased

susceptibility for venous thrombosis (120). The prevalence of factor V Leiden and

acquired APC resistance among women with recurrent miscarriage has been variably

reported to be either similar to or increased compared to controls

(121,122).

1.4.2.5 Plasminogen Activator Inhibitor 1 (PAI1)

Plasminogen activator inhibitor-1 is the principal inhibitor of tissue plasminogen

activator (tPA) and urokinase plasminogen activator (uPA), the activators of

plasminogen and hence fibrinolysis. Endothelial PAI-1 expression is modulated by a

4G/5G polymorphism in the PAI-1 promoter, which is 675 bp upstream from the start

site of transcription. Angiotensin II plasma levels also influence PAI-1 expression.

Homozygosity for the 4G allele of the PAI-1 gene increases the risk for pregnancies,

predisposing to prematurity, intrauterine growth retardation, miscarriage and stillbirth

(123).

1.4.2.6 Prothrombin

Prothrombin is the precursor to thrombin in the coagulation cascade. Thrombin is

required to convert fibrinogen into fibrin, which is the primary goal of the coagulation

cascade. G to A transition at nucleotide position 20210 in the 3’untranslated region of

the prothrombin gene has been associated with increased plasma prothrombin

concentrations, which increases risk for venous and arterial thrombosis (124). Though

Introduction



prothrombin mutation has been identified as a risk factor for RPL in some studies

(125), others have reported lack of association (126).

The various candidate genes of the vascular remodeling, their function and the effect

of their polymorphisms in RPL are summarized in Table 1.5.

1.4.3 Genetic polymorphisms in Xenobiotic metabolism

RPL is believed to be associated with maternal exposure to various xenobiotics like

environmental toxins and teratogens. Heavy metals such as lead and mercury, organic

solvents, alcohol, and ionizing radiation are environmental teratogens, and exposure

to these could contribute to pregnancy loss. Caffeine, cigarette smoking, and

hyperthermia are suspected teratogens, while the teratogenic impact of pesticides

remains unknown (132). Increased caffeine intake and deficient detoxification has

been positively related to an enhanced risk of RPL (133). Further, maternal exposure

to dioxin has also been associated with increase in fetal loss and reduction in birth

weight in experimental animal studies (134). However, there are only a few

epidemiological studies on the relation between the maternal dioxin and pregnancy

outcome in humans (135)

In addition to the lethal effects of the various xenobiotic substances described above,

oxidative stress also results in susceptibility to RPL. The increase in placental blood

circulation towards the end of first trimester results in an enhanced oxidative load,

which facilitates embryonic differentiation and development (136). However, an

excessive oxidative load along with inefficient antioxidant defense generates

abundant free radicals that could induce reproductive toxicity and thus prove lethal to

the embryo, resulting in RPL (137). Hence, the decidual or placental detoxification

system should be efficient and functional so as to protect the conceptus from

increased free radicals as well as xenobiotics.

Introduction


27

Candidate Genes Gene loci Polymorphism Gene function Effect of

Polymorphism

VEGF

(Papazoglou et al 2005) (127)

6p21 -2578 A/C Growth factor that is active

in angiogenesis and

vasculogenesis

Altered VEGF production

ACE

(Buchholz et al 2003) (123)

17q23 Insertion/ Deletion at

intron 16

Mediates conversion

angiotensin I to angiotensin

II .This enhances

vasoconstrictor activity of

angiotensin

Hypofibrynolysis

MTHFR

(Holmes et al 1999;

Unfried et al 2002) (128,129)

1p36 Codon 119 G to T

transition

Mediates conversion of

homocysteine to

methionine

Accumulation of

homocysteine

Factor V Leiden

(Townson et al 1997) (130) 1q21

G1691A

Produces factor V, which

converts prothrombin to

thrombin.

Resistance to activated

protein C

Table1.5: Polymorphisms in vascular networking and placental remodeling genes reported in RPL

Introduction


28

PAI1 (Buchholz et al 2003) (123) 7q21 4G/5G in the promoter Regulates fibrynolysis

Hypofibrynolysis

Prothrombin

(Hohlagschwandtner et al 2003)

(131)

11p11 G20210A

Blood clotting protein that is

needed to form fibrin

Increased prothrombin

concentration

Introduction



1.4.3.1 Phase I metabolism

Phase I metabolism the first enzymatic defense against foreign compounds is

mediated mainly by the CYP450 super gene family of enzymes, such as CYP1A1,

CYP1A2, CYP1B1 etc. In a typical phase I reaction, CYPP450 uses oxygen and

NADH as a cofactor to add a reactive group, such as a hydroxyl radical. As a

consequence of this step reactive molecules that are more toxic than the parent

molecule are produced (138).

1.4.3.2 Phase II metabolism

Phase II metabolism follows phase I activation, in which the reactive intermediates

are detoxified into water-soluble compounds that can be excreted through urine or

bile. Some of the phase II enzymes are acetyltransferases, glutathione S transferases,

uridine 5'-diphosphoglucuronosyl transferases, sulfotransferases, aldoketoreductases,

transaminases and hydrolases (139).

When the body is confronted with a high xenobiotic load, the phase I and/or phase II

enzymes involved in detoxifying this compound are induced, leading to xenobiotic

detoxification. The variations in metabolic activities in each phase or in the

coordination of the two phases affect the clearance of toxic metabolites. Most of the

phase I and phase II enzymes are polymorphic and the variants differ in their ability to

process the xenobiotics. Thus, inherited differences in the effectiveness of activation

and detoxification play a crucial role in RPL Fig 1.12.

1.4.3.3.1 Cytochrome P4501A1 (CYP1A1)

CYP1A1 is a major extra hepatic enzyme involved in the oxidation of the common

environmental toxicants such as polycyclic aromatic hydrocarbons (PAH) like

benzopyrene, polychlorinated biphenyls, etc. (140). Most of these aromatic

hydrocarbons bind to Aryl hydrocarbon (Ah) receptor. Following ligand binding, the

Ah receptor dimerizes with Ah receptor nuclear translocator, and there by acquires the

Introduction



ability to interact with xenobiotic response elements that enhance transcription of

genes encoding CYP1A1 (141, 142, 143). Oxygenation by P4501A1 serves as an

initial step in the metabolic conversion of the substrates to water-soluble metabolites

for excretion from the body. However, oxygenation of PAHs often generates

electrophilic arene oxide, dioepoxide and other reactive species which damage the

DNA and/or affect protein functions, leading to adverse effects.

1.4.3.3.2 CYP1A1 polymorphism

CYP1A1 gene mapped on 15q22-24 spans 10kb and consists of 7 exons. The CYP1A1

gene is polymorphic and four SNPs have been reported. First and the most important

polymorphism is T 3801 C substitution in the 3’ untranslated region (UTR) referred to

as m1 allele. As this polymorphism creates a restriction site for Msp1 enzyme, it is

also referred to as Msp1 polymorphism. Second, A 2455 G transition in exon 7

referred to as the m2 allele, leads to the substitution of Val for Ile at codon 462 in the

heme-binding region. Both the m1 and m2 allele have been reported to exhibit higher

catalytic activity (144).Third polymorphism is T 3205 C in intron 7 referred to m3

allele and is African-American specific. The fourth one is C 2453 A in exon 7

referred to as m4 allele, which leads to Thr to Asn substitution but its effect on

enzyme activity has not been clarified (145). These SNPs are represented in Fig 1.13.

Fig 1.12 – Xenobiotic metabolism highlighting the genes of interest

AngiogenesisAngiogenesisAngiogenesisAngiogenesis

Free

Radicals

Phase I

Reactive

Intermediates

Phase II

Water

Soluble

Compounds

Excreted

Activation Detoxification

CYP1A1 GSTM1

GSTT1

GSTP1

Introduction



1.4.3.4 Glutathione S Tranferase (GST) Family Enzymes

The GST family of enzymes are important members of phase II detoxification. They

catalyze the conjugation of a wide range of electrophilic substances to glutathione,

facilitating their elimination from the body. Moreover, GST enzymes also play an

important role in regulation of reduced glutathione levels, and thereby redox reserves

of the individual. Hence any decrease in the GST activity can lead to accumulation of

the products of phase I activity, which can cause severe damage. GSTs are present in

large amounts in many tissues including those of the genital tract, placenta and

deciduas (146, 147). They are expressed very early in the embryonic development and

hence believed to play a crucial role in female reproduction (148).

In humans eight distinct gene families encode GSTs. There are currently five putative

α class genes encoding GSTA1, GSTA2, GSTA3, GSTA4 and GSTw. The GSTP

class contains a single gene encoding the GSTP1 protein, and the θ class consists of

two genes encoding the GSTT1 and GSTT2 proteins and µ class consists of five genes

encoding GSTM1, M2, M3, M4 and M5. Of these GSTM1, GSTT1 and GSTP1

genes are frequently analyzed in disease susceptibility studies (147).

1.4.3.4.1 GSTM1 gene polymorphism

The GSTM1 gene belongs to the GSTµ class gene family, members of which are

located on a 100-kb gene cluster arranged as 5’-GSTM4-GSTM2-GSTM1-GSTM5

GSTM3–3’ at 1p13.3 (149). The GSTM1 gene consists of eight exons and is flanked

by two identical 4.2-kb regions as shown in Fig 1.13. The presence or absence of the

gene constitutes the polymorphism. The GSTM1 deletion caused by homologous

recombination involving the 5’ and 3’ 4.2kb repeats, results in 16kb deletion

containing the GSTM1 gene. The point of deletion has not been localized precisely

because of the high sequence identity repeats (149). Deletion of the GSTM1 gene

frequently affects both the alleles, resulting in the so-called null genotype GSTM-/-.

Distinct ethnic differences have been observed in the distribution of GSTM1 null

genotypes and the GSTM1 deletion frequencies range from 22.4% in South Indians

Introduction



(150), 28% in Africans (151), 33% in North Indians (152), 46.9% in Italians (153),

48.8% in Caucasians (154), 51% in Chinese (155) and 55.7% in Japanese (156).

1.4.3.4.2 GSTT1 gene polymorphism

GSTT1 gene is 8.1 kb in length and is part of the theta-class GST gene cluster at

22q11.2 (157). GSTT1 and GSTT2 are separated by approximately 50 kb. GSTT2 lies

head-to-head with a gene encoding the D-dopachrome tautomerase (DDCT). The

GSTT1 gene consists of five exons, and is flanked by two 18 kb regions, HA3 and

HA5, which are more than 90% homologous. The GSTT1 null allele (GSTT1-/-)

arises by homologous recombination of the left and right 403bp repeats, which results

in a 54kb deletion of the entire GSTT1 gene (Fig 1.14). The point of deletion has not

been precisely localized because of the sequence identity between the 403bp repeats.

GSTT1 gene is polymorphic in humans and the phenotypic absence of enzyme activity

is due to the deletion of the gene (158). GSTT1 deletion frequently affects both the

alleles and the deletion frequency exhibits ethnic differences. GSTT1 deletion

frequencies vary between 17% and 46% in different population (151, 152, 153, 154,

155, 156).

1.4.3.4.3 GSTP1 gene polymorphism

GSTP1 gene mapped to 11q13 is 2.8 kb long and encodes the GSTP1 protein of 209

amino acids (Fig 1.14). GSTP1 has a polymorphic site in exon 5, where an A to G

transition at nucleotide 313 results in an Ile105Val substitution in the substrate-

binding site of GSTP1 (159, 160, 161). Substitution of the less bulkier and more

hydrophobic valine results in substrate-dependent alterations of GSTP1 activity (160,

162). Although there is an increasing body of evidence suggesting an association

between the Ile105Val polymorphism of GSTP1 and cancer susceptibility, influence

of this polymorphism in RPL remains controversial. C to T transition at nucleotide

341 results in Ala114Val amino acid substitution. However, this polymorphism does

not appear to result in significant alteration in enzymatic function (160).

Introduction



GSTP1 gene is polymorphic in humans and the frequency of the valine allele exhibits

ethnic differences. The Val allele frequency is 3.1% in South Indians (150), 5.4 in

North Indians (152) and 13.3 in Caucasians (154).

1.4.3.5 Aryl hydrocarbon receptor nuclear translocator gene (ARNT)

polymorphism

ARNT belongs to a family of transcription factors that regulate a wide group of

biological functions including embryonic development and response to low oxygen

tension. ARNT dimerizes with hypoxia inducible factor 1 alpha (HIF1alpha) to

promote angiogenesis and trophoblast invasion in the first trimester of pregnancy. A

to C polymorphism in codon 511 of the ARNT gene, which results in the substitution

of Asn for Asp in the protein, has been implicated in RPL (163).

Allelic variants of GSTs that have impaired detoxification may enhance the rate of

genetic damage and thereby increase the susceptibility to reproductive toxicity, which

could lead to endometriosis, RPL or poor pregnancy outcome (164, 165, 166, 167,

168). Studies on GST gene polymorphisms and RPL have reported contrasting

findings and ethnic differences (166, 169, 170, 171).

The various candidate xenobiotic metabolizing genes, their function and the effect of

their polymorphisms in RPL have been tabulated (Table 1.6)

1.4.4 Genetic polymorphisms in DNA repair genes

In normal pregnancies, the early stages of development take place in a low oxygen

environment. This physiological hypoxia of the early gestational sac protects the

developing

fetus against the deleterious and teratogenic effects of oxygen

free

radicals. Various adaptive changes occur with advancing pregnancy to meet the

increasing demands for proper body functions of the mother to fulfill the requirements

of the fetus. One of the adaptive changes in the respiratory physiology from eight

Introduction


34

Candidate Genes Gene

loci

Polymorphism Gene function Effect of Polymorphism

CYP1A1

(Suryanarayana et al 2004)(171)

15q24 T to C in 3’ UTR

Oxidation of polycyclic

aromatic hydrocarbons

Increase in enzyme activity

CYP1B1

( Saijo et al 2004)(163)

2p21 432 C/G

Metabolic activation of

benzo[a]pyrene and estrogens 432 G (variant)

enzyme activity

was 3-fold higher than the 432C

CYP2D6

(Suryanarayana et al 2004) (171) 22q13

G1934A

Mixed-function oxidase

system involved in the

metabolism of xenobiotics

Enzyme with poor metabolism

GSTM1, GSTT1

(Zusterzeel et al 2000; (166)

Suryanarayana et al 2004) (171)

1p13, 22q11 Null

Conjugate reduced

glutathione to a number of

elecrophilic substances

including the products of

phase I metabolic

intermediates, thereby

facilitating their elimination

from the body

Loss of enzyme activity

GSTP1

(Zusterzeel et al 2000; (166)

Suryanarayana et al 2004) (171)

11q13 A313G

Ile105Val substitution alters

enzyme activity (101)

Table 1.6: Polymorphisms in xenobiotic metabolism genes reported in RPL

Introduction


35

Ah receptor

(Saijo et al 2004) (163)

7p15 554C/G

Regulates transcription of the

CYP450 family that

metabolizes many

carcinogens

Functional significance not well

established

NAT2

(Hirvonen et al 1996)(164)

8p22 M1, M2 and M3

M1- C481T and

T342C

M2 -C282T and

G590A

M3- G857A

Responsible for

the

acetylation that determines

whether individuals are slow

or fast acetylators of a number

of drugs and xenobiotics

Altered acetylation activity

Introduction



week onwards is the increase in ventilation from an initial 36% to a maximum of 50%

or more to meet the increasing demands of oxygen (172). This triggered aerobic

environment should primarily be responsible for raised oxidative stress in pregnancy.

Further, uncontrolled iron supplementation and environmental factors may add to the

oxidative stress. Above all, the feeble antioxidant defense, could lead to an

undesirable level of oxidative stress and oxidative damage of cellular and/or tissue

components. There is mounting evidence that oxidative stress or an imbalance in the

oxidant/antioxidant activity in utero–placental tissues plays a pivotal role in the

development of placental-related diseases (172). In miscarriage, development of the

placento–decidual interface is severely impaired leading to early and widespread

onset

of maternal blood flow and major oxidative degeneration.

This excess oxidative load and the subsequently induced DNA damage have to be

cleared and repaired. Cellular oxidants, such as free radicals and reactive oxygen

species (ROS) are also produced during natural metabolic process. ROS are highly

reactive and potentially damaging to the cells, because they directly affect

macromolecules and organelle functions. Damage to DNA by ROS results in single-

strand and double-strand breaks, apurinic and apyrimidinic sites and adduct

formation. In addition, ROS can catalyze the oxidative modification of proteins,

including enzymes involved in DNA repair (173). If the damage is recognized by the

cell machinery, responses like cell cycle arrest may occur to prevent replication in the

presence of genetic errors (174). Integrity of the so-damaged DNA is typically

restored as a consequence of DNA-repair enzymes, the normal function of which is

important for maintaining genomic integrity (175).

In addition to oxidative stress, transplacental exposure to carcinogenic air pollutants

such as polycyclic aromatic hydrocarbons might cause DNA damage by forming

chemical-DNA adducts. This suggests that DNA repair capacity is essential for the

maintenance of pregnancy, but little is known about the direct effect of DNA repair

capacity on RPL. Complex pathways involving numerous molecules perform DNA

repair. Cells with unrepaired DNA damage either undergo apoptosis or unregulated

growth to malignancy. Integrity of the damaged DNA is typically restored as a

consequence of DNA repair enzymes, the normal function of which is important to

maintain genomic integrity.

Introduction



1.4.4.1 DNA repair pathways

DNA repair pathways that operate on specific types of DNA damage are: Base

excision repair, Nucleotide excision repair, Mismatch repair, Homologous

recombination and non-homologous end joining.

1.4.4.1.1 Base excision repair

Base excision repair removes simple base modifications, including single-strand

breaks, oxidative DNA damage and non-bulky adducts. The damaged base is removed

by base-specific DNA glycosylases. The abasic site is excised by the endonuclease

action of apurinic/apyrimidinic endonucleases (APEX). DNA synthesis is then

catalyzed by DNA polymerase using the other strand as the template. Following this,

the DNA strands are ligated (176). Molecules involved in the polymerization and

ligation of base excision repair include, polynucleotide kinase, XRCC1 and DNA

ligase (Fig 1.15).

1.4.4.1.2 Nucleotide excision repair

It removes larger lesions, such as pyrimidine dimers, photo-adducts, large chemical

adducts and cross-links which often result from environmental damage, including UV

radiation and external carcinogens. The nucleotide excision repair pathway involves

four steps (Fig 1.16). Damage recognition by a complex of bound proteins including

XPC and XPA; unwinding of the DNA by the TFIIH (transcription Factor II H)

complex that includes XPC, XPA, XPD and XPB; Removal of the damaged single-

stranded fragment (usually about 27–30 bp) by molecules including ERCC1 and XPG

complex; and Polymerization or synthesis of the strand by DNA polymerase and

ligation.

As genetic polymorphisms in DNA-repair enzymes influence DNA adduct levels

(177, 178, 179) DNA repair capacity may be associated with the risk of RPL. The

present study focused on the DNA repair genes, X-ray repair cross complementing

group 1 (XRCC1) and xeroderma pigmentosum group D (XPD). Association of these

Introduction



polymorphisms in XRCC1 and XPD genes have been shown in various cancers like

lung (180, 181), head and neck (182, 183) and breast (184).

1.4.4.1.3 XRCC1 gene polymorphism

The XRCC1 gene mapped to 19q13.2–13.3, spans 32.25 kb encoding XRCC1 protein

(Fig 1.17). XRCC1 involved in base excision repair, appears to play a scaffolding

role in bringing together a complex of DNA repair proteins, including poly (ADP-

ribose) polymerase (PARP), DNA ligase 3 (LIG3), polynucleotide kinase, AP

endonuclease and DNA polymerase β (185). XRCC1 is believed to form complexes

with DNA polymerase β at its NH2 terminus and with DNA ligase III via the breast

cancer COOH-terminus (BRCT) domain to repair the gaps during base excision

repair.

Two common single nucleotide polymorphisms (SNPs) that have been reported in the

coding region of XRCC1 gene are C to T transition at position 26304 in exon 6 which

leads to an Arg to Trp substitution at codon 194 and G to A transition at position

28152 in exon 10 which leads to an Arg to Gln substitution at codon 399 (Fig 1.17).

These polymorphisms involve an amino acid change at the evolutionarily conserved

regions and could alter the XRCC1 function.

1.4.4.1.4 Xeroderma pigmentosum group D (XPD) gene polymorphism

XPD gene also referred to as ERCC2, mapped to 19q13.3 is comprised of 23 exons,

spans about 54 kbp (Fig 1.17). XPD gene encodes the 86.9 kDa XPD protein which is

a component of the transcription factor TFIIH. XPD possess single strand DNA-

dependent ATPase activity and 5’-3’ helicase activity, thus participating in DNA

unwinding during NER and transcription (186)

Two common SNPs reported in the XPD gene are G to A transition in exon 10, which

results in Asp to Asn substitution at codon 312, and C to A transversion at 35931

nucleotide of exon 23, which results in a Lys to Gln substitution at codon 751.

Mutations in XPD gene can completely prevent DNA unwinding and excision of the

Introduction



affected strand (187). David Baebes et al 2001 (181) reported that individuals with the

XPD 751 Lys/Lys genotype have a 7-fold increased risk of suboptimal DNA repair.

Qiao et al 2002 (187) observed individuals with 751 Gln/Gln to exhibit sub-optimal

DNA repair to remove UV photoproducts compared to the XPD 751 Lys/Lys

genotypes.

Since the XRCC1 and XPD genes play an important role in DNA repair mechanisms,

the variation in these genes might affect maintenance of pregnancy and thus may be

associated with RPL. Currently, to the best of our knowledge, there are no studies on

DNA repair gene polymorphisms in RPL. The present study is the first of its kind to

analyze the role of polymorphisms in DNA repair genes as a susceptibility marker for

RPL.

1.4.5 Other Genetic polymorphisms in RPL

1.4.5.1 Progesterone Receptor Gene

Progesterone is required for the maintenance of pregnancy and treatment with

progesterone supplementation prevents abortions in some individuals. Three linked

SNPs have been detected in the progesterone receptor gene – exon 1: G 1031 C; Ser

344 Thr, exon 4: G 1978 T; Leu 660 Val, exon 5: C 2310 T; His 770 His (188).

1.4.5.2 Cytochrome P450c17 alpha (CYP 17)

The CYP17 gene encodes the enzyme cytochrome P450c17a, which mediates both

17a-hydroxylase and 17, 20-lyase activity in the steroid biosynthesis pathway. T to C

polymorphism in the 5’ promoter region of CYP17 results in a variant allele (A2).

Women with the CYP17A2 allele have been reported to exhibit increased risk of RPL.

(189).

Introduction



1.4.5.3 p53

p53, a tumour suppressor gene encodes a multifunctional transcription factor that is

activated by stress stimuli including DNA damage and hypoxia. It is a well-known

factor regulating apoptosis in a wide variety of cells and also plays a critical role in

angiogenesis (190). In addition, p53 has recently been reported as a potential mediator

of pregnancy by estrogen and progesterone activation (191). A common sequence

polymorphism in p53 is located within the proline-rich domain encoding either

proline or arginine at position 72. The Proline allele has been reported to increase the

risk of RPL (192).

From the literature it is evident that polymorphisms in genes regulating

immunomodulation, angiogenesis, vascular network, xenobiotic detoxification and

DNA repair have an impact on the individual susceptibility to RPL. Further, the

contribution of genetic polymorphisms to the risk of RPL is dependent on the

population studied, as well as environmental and dietary factors that influence the

population. Epidemiological studies have shown that, polymorphisms in the genes

regulating initiation and maintenance of pregnancy attribute to the risk of RPL.

However, these study reports are inconsistent. Moreover, studies on association of

genetic polymorphisms in patients with RPL is very limited in the Indian population,

especially in South Indians. Thus, identification of susceptibility markers is essential

to determine the genetic predisposition to RPL.

For a successful pregnancy outcome, the first step is the modulation of the maternal

immune system to tolerate the semi allogenic fetus. HLA G and KIR alleles have been

proposed to play a major role in this process. The next stage is the establishment of

vascular network and proper angiogenesis between the growing fetus and the uterine

wall of the mother. This process is regulated by the VEGF, MTHFR and ACE gene

products. The excess oxidative stress induced during angiogenesis may prove lethal to

the developing embryo. This surplus oxidative load and the subsequent DNA damage

that could be induced have to be cleared and repaired. This function is mediated by

the enzymes encoded by detoxifying genes such as CYP1A1, GSTM1, GSTT1, GSTP1

and the DNA repair genes like XRCC1 and XPD. Hence the present study adopts a

Introduction



multigeneic approach and hypothesizes that polymorphisms in genes that alter the

normal function of these pathways might result in an increased susceptibility to

RPL.The study proposition is represented in Fig –1.18.

Introduction



1.5 Aim and Objectives of the Study

The main aim of the study is to determine the association of the immunological and

genetic factors in the risk of unexplained recurrent pregnancy loss in South Indian

population. In order to investigate the role of immunological factors, gene

polymorphisms were analysed in the spouses. Whereas to assess the other proposed

genetic factors that play a role in embryonal development, only the maternal gene

polymorphisms were examined.

The specific objectives are:

1. To examine association between unexplained recurrent pregnancy loss and

immunomodulatory genes, with specific reference to HLA G and the KIR

(i) To investigate the role of HLA G and KIR polymorphisms in women

with unexplained RPL compared with the control women.

(ii) To determine the role of paternal HLA G and KIR polymorphisms in

RPL.

(iii) To determine the association of sharing of the alleles between the

spouses in the outcome of pregnancy with specific reference to the

HLA G and KIR repertoire.

(iv) To explore the role of anti-paternal cytotoxic antibodies (APCA) in

RPL.

2. To determine the risk of RPL attributed by polymorphism in genes regulating

a. Placental vascular network, with specific reference to

(i) Vascular Endothelial Growth Factor (VEGF) gene

(ii) Angiotensin Converting Enzyme (ACE) gene

(iii) Methylene tetra hydrofolate reductase (MTHFR) gene

b. Xenobiotic metabolism, with specific reference to

(i) Cytochrome P4501A1 (CYP1A1) gene

Introduction



(ii) Glutathione-S transferase µ (GSTM1) gene

(iii) Glutathione-S transferase pi (GSTP1) gene

(iv) Glutathione-S transferase θ (GSTT1) gene

c. DNA repair pathway, with specific reference to

(i) X-ray repair cross-complementary group 1 (XRCC1) gene

(ii) Xeroderma pigmentosum group D (XPD) gene

3. To correlate the genotypes with age and the number of pregnancy loss at

diagnosis.

Introduction


South Indian population 44

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