Chapter Chapter ---- IIII Introduction and Review of …shodhganga.inflibnet.ac.in/bitstream/10603/2325/9/09... · 2015-12-04 · Chapter I: Introduction and Review of Literature

Chapter Chapter Chapter Chapter ---- IIII

Introduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of Literature

Chapter I: Introduction and Review of Literature

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Chapter - I

Introduction and Review of Literature

The Darwinian fitness of a population is directly related to its reproductive

fitness. Women and children are the greatest asset of a nation as the well being and

development of the nation is determined and directed by the maternal and child

health since they are responsible for the complete physical, mental and social well

being of the population. One of the major aims of the Reproductive and Child

Health approach underlines that the women are able to go through pregnancy and

child birth safely and that the outcome of pregnancy is successful in terms of

maternal and fetal survival. Management and treatment of infertility and various

fatal pregnancy complications poses a major burden on the society and is a

challengeable task for researchers world over. Only 30% of all human conceptions

are reported to result in a live birth (Macklon et al., 2002) and others leading to

pregnancy failure; this not only causes physical harm but also creates a mental

trauma in a family. Thus there is an urgent need to understand the complex

pathogenesis and genetics of unexplained pregnancy loss for their management.

1.1. Recurrent Pregnancy Loss

World Health Organization (WHO) defines the spontaneous abortion as “the

expulsion or extraction of a fetus weighing 500 g or less (approximately equal to

20-22 weeks of gestation) or another product of gestation of any weight and

specifically designated irrespective of gestational age whether or not there is

evidence of life” (WHO, 1976). Most of the spontaneous abortions or miscarriages

occur in the first trimester and affect about 15% of all recognized pregnancies (Ford

and Schust, 2009).


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Recurrent Pregnancy Loss (RPL) or Habitual Abortion is defined as “the occurrence

of three or more clinically detectable pregnancy losses” (Stirrat, 1990), prior to 20

weeks of gestation (Sierra and Stephenson, 2006). In simple words, the cases

suffering from recurrent pregnancy loss are the women who show a history of three

or more sequential first trimester pregnancy losses without any live issue in between

or thereafter (Stevenson, 1996). Epidemiologic studies have revealed that 0.34 to

2% of women who conceive experience recurrent pregnancy loss (Christiansen et

al., 2005; Ford and Schust, 2009). Thus it becomes extremely important to

understand the etiology caused by various factors in manifestation of recurrent

pregnancy loss.

1.1.1. Causes of Recurrent Pregnancy Loss

The causes of the recurrent pregnancy loss are mainly categorized into fetal

and maternal causes. Fetal causes are the abnormalities within the fetus itself which

does not allow it to implant and grow properly in the mother’s womb like

chromosomal abnormality in the fetus itself or some congenital malformation. The

maternal causes refer to problems within the uterine environment that does not

allow the fetus to grow properly and is thus aborted. Many researchers have

described various genetic (single gene mutations, polygenic and cytogenetic factors)

and non-genetic (congenital uterine abnormalities, infections, hormonal imbalances,

nutritional deficiencies and psychological factors) causes (for review see Meka and

Reddy, 2006). In the following a brief description of some of these causes is given.

Uterine anatomical defects

Distortion of the uterine cavity may be found in approximately 10 to 15% of

women with recurrent pregnancy losses. Congenital uterine abnormalities include a

uterine septum, double uterus and a uterus in which only one side has been formed.

On the other hand, Asherman’s syndrome (scar tissue in the uterine cavity), uterine

fibroids, uterine leiomyomata and possibly uterine polyps are the acquired


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abnormalities that may also cause recurrent miscarriages. Women with recurrent

pregnancy loss have an anatomic abnormality of the uterus as the primary reason

mainly because of the incompetent cervix (weak cervix) which results in the mid

trimester loss of pregnancy.

Chromosomal aberrations

About half of first trimester pregnancy losses occur due to chromosomal

abnormalities, such as a missing or a duplicate chromosome. Fetus carrying

chromosomal abnormalities is expelled naturally in the early stages of pregnancy as

it is not viable. Translocation is the most common inherited chromosomal

abnormality. Balanced reciprocal or robertsonian translocations in couples which

otherwise do not have lethal effect on the individual but result in the chromosomal

abnormalities in the fetus at the time of formation of daughter cells, also result in

the miscarriage at the early stages of pregnancy. Additional structural abnormalities

associated with RPL include inversions, insertions and mosaicisms.

Hormonal and metabolic disorders

These constitute about 10 to 20 % causes of RPL. Progesterone, a hormone

produced by the ovary after ovulation, is necessary for a healthy pregnancy. There

is controversy about whether low progesterone levels, often called luteal phase

deficiency, may cause repeated miscarriages. Thyroidism, diabetes mellitus,

inadequate progesterone, insufficient luteal progesterone and increased androgens

due to polycystic ovary syndrome either result in infertility or early pregnancy loss.

Poorly controlled diabetes increases the risk of miscarriage. Women who have

insulin resistance, such as obese women and many who have polycystic ovarian

syndrome, also have higher rates of miscarriage.


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Haematological problems

These include thrombophilia and hemorrhage occurring in the placenta

leading to either miscarriage or initiating abruption or restricted blood flow finally

leading to fetal death, especially in second half of pregnancy. The heritable

thrombophilias most often linked to RPL include hyperhomocysteinemia resulting

from MTHFR mutations, activated protein C resistance associated with factor V

Leiden mutations, protein C and protein S deficiencies, prothrombin promoter

mutations, and antithrombin mutations. Acquired thrombophilias associated with

RPL include hyperhomocysteinemia and activated protein C resistance (Ford and

Schust, 2009).

Although definite causative links between these heritable and acquired

conditions have yet to be solidified, the best available data suggest testing for factor

V Leiden mutation, protein S levels, prothrombin promoter mutations,

homocysteine levels, and global activated protein C resistance, at least in white

women (Rey et al., 2003; Kovalevsky et al., 2004; Robertson et al., 2006). Kumar et

al. (2003) showed an association of MTHFR gene mutation in women with

unexplained RPL in India. Similarly, Mukhopadhyay et al. (2009) reported a

statistically significant positive association between inherited thrombophilia with

respect to MTHFR C677T and FVL mutations in recurrent abortions in north Indian

population.

Immunological factors

Immunological factors are the most poorly understood causes. They are

mainly categorized into autoimmune and alloimmune disorders. Autoimmune

causes are the one where mother’s immune system attacks her own organs and

tissues e.g. anti-phospholipid syndrome (APS) resulting in RPL, fetal death and

thrombosis (Wilson et al., 1999). APS is classically defined as a triad of recurrent

pregnancy loss, thrombosis and autoimmune thrombocytopenia (decreased platelet

concentration). Alloimmune disorders on the other hand are the ones where the


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mother’s immune system attacks tissues considered to be foreign. In pregnancy, the

placenta and growing embryo are not entirely "self" but rather are a result of both

the maternal and paternal genetic heritages (referred to as a semi-allograft). The

placenta (and pregnancy) has a "privileged" relationship with the pregnant woman

that allows for it to escape rejection. The mechanism for this privilege is not known.

There have been several interesting and complex theories attempting to describe

how the normal pregnancy achieves its privileged status in the maternal uterus.

Meka and Reddy (2006) described the role of human leukocyte antigens (HLA) in

pregnancy loss. Excess sharing of HLA between spouses has been considered by

some to be a mechanism leading to maternal hypo- responsiveness to paternal

antigens encountered in pregnancy and therefore subsequent miscarriage (Beer et

al., 1981).

Uterine infections

Numerous organisms have been implicated in the sporadic cause of

miscarriage, but common microbial causes generally have not been confirmed. The

major organisms which can lead to sporadic pregnancy loss are Listeria

Monocytogenes, Toxoplasma Gondii, Rubella, Herpes Simplex, Measles,

Cytomegalovirus and Coxsackievirus. The proposed mechanisms for infectious

causes of pregnancy loss include: (a) direct infection of the uterus, fetus, or

placenta, (b) placental insufficiency, (c) chronic endometritis or endocervicitis, (d)

amnionitis and (e) infected intrauterine device.

Studies have been done to determine the extent of maternal infection with

respect to Toxoplasma Gondiiin reproductive disorders in India (Oumachigui et al.,

1980; Pal and Aggarwal, 1979). However, the role of infectious agents in recurrent

pregnancy loss is less clear, with a proposed incidence of 0.5 (Stevenson, 1996) to

5% (Fox-Lee and Schust, 2007). The particular infections speculated to play a role

in RPL include mycoplasma, ureaplasma, Chlamydia Trachomatis, L.

Monocytogenes and HSV (Summers, 1994) A number of maternal infections can


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lead to a single pregnancy loss, including Listeriosis, Toxoplasmosis, and certain

viral infections such as Rubella, Herpes Simplex, Measles, Cytomegalo virus and

Coxsackie virus. Malaria, syphilis and brucellosis can also cause recurrent abortion.

However, there are no confirmed studies to suggest that specific infections will lead

to recurrent pregnancy loss in humans.

Other factors

The chance of the miscarriage increases as the woman ages. After age 40

more than one-third of the pregnancies result in miscarriages as most of the

embryos have an abnormal number of chromosomes. Ovarian age, life style factors

and psychological factors are known to be associated with termination of pregnancy

but they being the cause of recurrent pregnancy loss are not well established.

Increasing evidence also suggests that abnormal integrity (intactness) of sperm

DNA may affect embryo development and possibly increase miscarriage risk.

Although several etiological factors have been established, still about 40 -

50% cases of recurrent pregnancy losses (RPL) remain unexplained (Stevenson,

1996; Quenby et al., 2002; Daher et al., 2003) and these might be explained by the

immunological factors (Shormilla and Knapp, 2000). It has been suggested that in

some women this may be due to an exaggerated maternal immune response to the

fetus (Babbage et al., 2001). The normal pregnancy is known to be associated with

the modulation of mother’s immune system in order to accept the conceptus, which

goes against the laws of immunology; whereas in case of spontaneous abortion, the

laws of immunology appear to be dominant over that of obstetrics.

1.2. Immunology of Pregnancy

The implanting embryo inherits its antigens from both father and mother and

is thus half-foreign to mother’s body. In other words, embryo acts as an alllograft to

the mother’s body and yet unlike a mismatched organ transplant it is not normally

rejected by the mother’s immune system and survives normally in the mother’s


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womb during the entire gestational period in the case of normal successful

pregnancy. This fact has provoked the researchers to reveal first how the mother’s

immune system reacts to the implanting embryo? That is, does the mother’s

immune system recognize the implanting embryo as foreign? And if it does, how

the placenta protects itself from the mother’s immune system to survive

successfully? Or in other words, how the implanting embryo suppresses the

mother’s immune system so that it is not treated as foreign and thus not rejected by

the mother’s body?

Pregnancy in humans presents a paradox for the mother's immune system as

the mechanisms which are essential to protect her from infection have the potential

to destroy her antigenically foreign fetus. The maternal decidua is comprised

principally of immune cells and it is into this tissue that the fetal trophoblast must

invade to establish the placenta. Both local and systemic nonspecific suppressor

mechanisms have been described which may down-regulate maternal immune

responses without significantly impairing the ability to fight infections, but there is

little evidence to suggest that specific blocking factors (antibodies and suppressor

cells) play an essential role. The placental barrier restricts the traffic of cytotoxic

cells to the fetus, and cytotoxic antibodies are removed by the placenta before they

reach the fetal circulation. Thus a combination of immune adaptations ensures the

success of the pregnancy (Sargent, 1993).

The immunological relationship between the mammalian fetus and its

mother during pregnancy has been considered similar to that between a transplanted

allograft and its recipient ever since Medawar (1953) first proposed the concept of

the 'fetus as an allograft' in the early 1950s. Based on this analogy, it has been

assumed that implantation of the fetal placenta in the uterus would be controlled

similarly by a maternal immune response mediated by T-cells recognizing

paternally-derived alloantigens expressed by the placenta. The cellular and

molecular basis of this local immune interaction between the fetal placenta and

maternal uterus has been the focus of intense research interest. Since aberrant


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implantation can cause a variety of clinical problems, including miscarriage,

intrauterine growth retardation and pre-eclampsia, an understanding of the

immunological mechanism by which this process is controlled could lead to the

development of regimens to improve fetal growth and development (King and

Loke, 1999).

Implantation is a process that involves development, attachment and invasion

of the blastocyst into the endometrium. Successful implantation requires appropriate

communication between the embryo and maternal endometrium. There is evidence

to suggest that cytokines produced by the maternal endometrium and the developing

embryo play a crucial role in this signalling process (Kauma, 2000). Chaouat et al.

(2002) suggested that the materno-foetal relationship is not simply maternal

tolerance of a foreign tissue, but a series of intricate mutual cytokine interactions

governing selective immune regulation and also the control of the adhesion and

vascularization processes during this dialogue.

Immunological mechanisms induced by T cells may play an important role

in preimplantation and embryo development in implantation process and in the

phenomenon of fetal allograft tolerance. Different cytokines produced by T cells

acting in concert are required to create a suitable microenvironment for the

preimplantation and embryo development and for the maintenance of pregnancy. T

cells could work in parallel with other cells present in the decidua and cumulus

suggesting a complex network of hormones, cytokines and cells (Piccinni, 2002).

Maternal tolerance of the fetal allograft could be the result of the integration

of numerous mechanisms promoted by different cells present in the decidual

macrophages. Dendritic cells which are found in close association with T

lymphocytes are the most potent activators of T lymphocyte responses and could

play a sentinel function for the immune system initiating antigen-specific T cell

responses to fetal antigens. T cell cytokines produced in response to fetal molecules

could have a role in the maintenance or in the failure of pregnancy (Piccinni, 2005).


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Recognition of the trophoblast brings about an inflammatory reaction which is the

initial phase of graft rejection. The numerous cytokines that are produced in this

initial phase allow decidualization to occur and for the embryo to implant when it

has reached an adequate stage of evolution. Rapidly, immunosuppressant

mechanisms stop this rejection reaction which if not stopped can cause the

pregnancy to end. There is a delicate equilibrium between the different cytokines,

those favorable to pregnancy and those damaging to pregnancy. The trophoblast

which is resistant to factors which would cause rejection protects the fetus

particularly if its growth is helped along by certain cytokines. On the other hand,

other cytokines are prejudicial to the growth of the trophoblast and activate certain

cytotoxic cells which become aggressive. The maternal immune system and the

endocrine system work together to maintain this cytokine network which if

destabilized leads to certain pathological situations. Disturbances can be due to poor

maternal recognition particularly if the trophoblast does not give out good antigens,

or if the mother is genetically programmed not to respond although the disturbance

can come from external factors such as certain infections (Vinatier and Monnier,

1993).

Piccinni (2006) reviewed the literature to understand the possible role of T

cells in successful pregnancy and in unexplained recurrent abortion. The functions

exhibited by Th1 and Th2 cells have suggested, perhaps in a simplistic way, that

Th1-type cytokines, which promote allograft rejection, may compromise pregnancy,

whereas the Th2-type cytokines, by inhibiting Th1 responses, promote allograft

tolerance and therefore may improve fetal survival. However, Th1 cytokines are not

always detrimental for pregnancy development. Th1 cytokines, depending on their

time of expression, stage of gestation and relative concentrations, could have a

positive role in successful pregnancy. Other cytokines (LIF, M-CSF) produced by T

cells seem to be important for the maintenance of pregnancy. Hormones present in

the microenvironment of the decidual T cells could be responsible, at least in part,

for the cytokine profile of the T cells. Indeed, progesterone is a potent inducer of


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Th2-type cytokines (e.g. IL-4 and IL-5), LIF and M-CSF production by T cells,

whereas relaxin induces T cells to produce IFN-gamma. Of course, the success of

pregnancy depends on many mechanisms induced by different types of cells and

Th2 cells could be one of those (Piccinni, 2006).

Clinical and experimental evidence has indicated that the maternal immune

response is biased toward antibody production and away from cell-mediated

immunity during pregnancy, especially in the vicinity of the fetal-placental unit.

Because antibody responses are often associated with the Th2 cytokine pattern, this

suggests that Th2-type cytokines might predominate locally in the regulation of the

maternal immune response. In order to test this hypothesis, Lin et al. (1993)

examined the local and distal release of cytokines during murine pregnancy using

ELISA assays. They reported that the Th2-specific cytokines IL-4, IL-5, and IL-10

were readily detectable in cell supernatants derived from fetal-placental units in all

three trimesters of gestation. These cytokines were detected in lysates of freshly

isolated, day 12 decidual and placental cells and in supernatants as early as 15 min

after the beginning of culture. The presence of functional IL-10 was confirmed by

specific bioassay. IL-10 mRNA was localized to the decidua at day 6 of gestation

by in situ hybridization. IFN-gamma (Th1 specific cytokine) was also found in the

supernatants from the first trimester of pregnancy, but was barely detectable in the

second, and undetectable in the third trimester. Cytokine expression was

consistently detected in samples from individual mice. None of these cytokines was

produced by unstimulated spleen or mesenteric lymph nodes from pregnant mice.

IL-4, IL-10, and IFN-gamma were produced by Con A-stimulated spleen cells from

virgin mice, but in ratios opposite to those found in the placenta. These observations

indicate that Th2-specific cytokines are normally produced at the maternal-fetal

interface. The continuous presence of IL-4, IL-5, and IL-10, with early and transient

expression of IFN-gamma, can provide a molecular basis for the antibody/Th2-like

bias of the maternal immune response during pregnancy (Lin et al., 1993).


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A special interaction is established during pregnancy between the maternal

immune system and fetal cells to allow the survival and the normal growth of the

fetus. Fetal cells expressing paternal alloantigens are not recognized as foreign by

the mother because of an efficient anatomic barrier and a local immunosuppression

determined by the interplay of locally produced cytokines, biologically active

molecules and hormones. A special balance between TH1 and TH2 lymphocytes

has also been observed at the fetal-maternal barrier that contributes to control the

immune response at this level. An important role is played by trophoblast cells that

act as a physical barrier forming a continuous layer and exert immune-modulatory

function. Trophoblast cells have also been shown to express regulators of the

complement system and to down regulate the expression of HLA antigens.

Dysfunction of these cells leads to morphological and functional alterations of the

fetal-maternal barrier as well as to hormonal and immune imbalance and may

contribute to the development of pathologic conditions of pregnancy, such as

recurrent spontaneous abortions (Bulla et al., 2004).

Pregnancy is an intriguing immunologic phenomenon. In spite of genetic

differences, maternal and fetal cells are in close contact over the whole course of

pregnancy with no evidence of either humoral and/or cellular immunologic

response of mother to fetus as an allotransplant. The general opinion is that the

fundamental protective mechanism must be located locally at the contact-plate,

between the maternal and fetal tissues. Immunologic investigations proved the

presence of specific systems which block the function of antipaternal maternal

antibodies, as well as formation of cytotoxic maternal T-cells to paternal antigens.

The protective mechanisms have been reported to be coded by genes of MCH

region, locus HLA-G (Milasinović, 2002).

Pregnancy is an immunological balancing act in which the mother's immune

system has to remain tolerant of paternal major histocompatibility (MHC) antigens

and yet maintain normal immune competence for defense against microorganisms.

Another major factor proposed by Sargent (1993) that appears to prevent the


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rejection of the trophoblast is its expression of HLA-G, a non-polymorphic

transplantation antigen. The placenta separates fetal and maternal blood and

lymphatic systems and it is fetal trophoblast that plays the major role in evading

recognition by the maternal immune system. Trophoblast cells fail to express MHC

class I or class II molecules and the extravillous cytotrophoblast cells strongly

express the non-classic MHC gene encoding HLA-G, which may down regulate

natural killer (NK) cell function. In addition, the trophoblast expresses Fas ligand,

thereby conferring immune privilege: maternal immune cells expressing Fas will

undergo apoptosis at the placenta/decidua interface. A third protective mechanism

exploited by the trophoblast is the expression of the complement regulatory proteins

CD46, CD55, and CD59. Uterine decidual and placental cells produce a huge array

of cytokines which, in part, contribute to the deviation of the immune response from

Th1 to Th2. This may leave the mother more open to infection whose control is

Th1-dependent, but increased production of Th1 cytokines has been linked to

spontaneous abortion and small-for-dates babies. This bias in cytokines and

hormonally mediated effects on the thymus and on B cells may also contribute to

the suppression of autoimmune responses and changes in circulating and local T-

cell subsets in pregnancy (Weetman, 1999).

To summarize, various researchers have proposed that in order to prevent the

rejection of implanting embryo, the mother’s immune system gets modulated in

such a way that it helps fetal allograft to survive. However, in cases of pregnancy

loss, deregulation of the mother’s immune system could be responsible for the

failure where the implanting embryo is recognized as foreign and is thus rejected by

the mother’s immune system, resulting in spontaneous abortion. Some of the

biological pathways revealed involved in the modulation of mother’s immune

system during implantation are listed below.


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1. Trophoblasts, decidual cells and cells of lymphnodes draining the uterus at

the time of implantation, suppress the mother’s immune responses (Clarke et

al., 1984; Bobe et al., 1986).

2. There is a lack of strong maternal cell mediated anti-fetal immunity and a

dominant humoral response (Mosmann and Coffmann, 1989; Wegmann et

al., 1993; Romagnani, 1994; Voison and Raghupathy, 1995; Mosmann and

Sad, 1996).

3. Semen contains TGF-β, which helps maternal immune system to tolerate

molecular signatures by altering the production of inflammatory cytokines

(Tremellen et al., 2000).

4. HLA released from the trophoblasts into mother’s blood stream seems to

protect them from the attack. Soluble HLA-G makes certain type of T-cells

which attack fetal cells bearing father’s antigens to commit suicide (Fournel

et al., 2000).

5. CRH secreted by both implanting embryo and the lining of uterus stimulate

trophoblasts to produce fas-ligand which binds to cell surface receptor that

triggers cell death of mother’s T cells.

6. Previous exposure to a fetus carrying a particular suite of paternal genes

makes the immune system more likely to bear first born’s subsequent

siblings (Pearson, 2002).

7. Appropriate regulation of classical and nonclassical MHC class I genes in

the trophoblast cells that form the outermost layer of the placenta is critical

for maternal immunological acceptance of the fetal-placental allograft

(Davies, 2007).

8. NK cells flooding the uterus at the time of implantation carry receptors that

interact with HLA-C and HLA-E on surface of trophoblasts, triggering he

production of particular cytokines that help trophoblasts to invade or limit

the extent to which it invades (Mofett-King, 2002).


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9. Anti-inflammatory cytokines dominate during pregnancy and act

antagonistically to pro-inflammatory cytokines to promote placentation and

embryonic development (Raghupathy, 1997).

1.2.1. Role of Natural Killer cells

The fetus is considered to be an allograft that paradoxically survives

pregnancy despite the laws of classical transplantation immunology. There is no

direct contact of the mother with the embryo, but only with the extraembryonic

placenta as it implants in the uterus. Convincing evidence of uterine maternal T-cell

recognition of placental trophoblast cells has been found, but instead, there might be

maternal allorecognition mediated by uterine natural killer cells that recognize

unusual fetal trophoblast MHC ligands (Moffett-King, 2002).

During implantation, maternal tissues are invaded by fetal trophoblasts

expressing HLA-G, a trophoblast-specific variant of HLA Class I antigens.

Recognition of HLA-G stimulates uterine natural killer cells to cytokine production,

by which an intrauterine immunosuppression is established. Development, growth

and differentiation of placenta are regulated by the cytokines produced. Uterine

leukocyte population and expression of cytokine receptors in placental tissues varies

throughout gestation, and the complex interplay between trophoblasts and uterine

cells, involving a number of cytokines, cytokine receptors, adhesion molecules,

enzymes and hormones, changes with gestation. Some cytokines, such as tumor

necrosis factor and interleukin-1, may threaten the reproductive process and fetal

well-being in high doses. A tight regulation of cytokine activities is probably

obtained by the observed up regulation of endogenous cytokine buffer mechanisms

in pregnancy. The reproductive success and phenomenon like implantation,

placental growth and development, maintenance of pregnancy and delivery, appear

to rely on complex, gestational age related interplay between cells of fetal origin

and the maternal immune system (Austgulen and Arntzen, 1999).


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Surprisingly, some evidence suggests that implantation might involve

predominantly a novel allogeneic recognition system based on natural killer cells

rather than T-cells (Loke et al., 1995). Conventionally it is considered that type 1

and type 2 cytokines are secreted only by CD4+ Th cells (Sargent at al., 2006). It is

well recognized that the two types of cytokines are not only produced by CD4+ T

cells, but also by CD8+ cytotoxic T (Tc) cells, NK cells and NKT cells (Carter et

al., 1995; Peritt et al., 1998). Therefore, the concept of “type 1/type 2” balance has

been extended largely by demonstrating that cytotoxic T cells, NK cells and NKT

cells can also play important roles in their cytokine secretion profiles. Recently,

Borzychowski et al. (2005) used the surface marker of IL-18 receptors for type 1

cells and ST2L for type 2 cells to distinguish all of the T cell and NK cell

populations from total lymphocytes. Their study showed that the type 2 shift during

pregnancy was predominantly in the NK (CD56+CD3-) cells and NKT

(CD56+CD3+) cells instead of in the T-helper cells or cytotoxic T cells. So they

proposed that innate immune system, involving NK cells and NKT cells may be

predominant population of the peripheral blood in pregnancy, which has challenged

the concept of Tom Wegmann’s first hypothesis that that fetal survival depends on a

bias of maternal immune responses towards T-helper Th2 immunity and the

inhibition of cytotoxic Th1 responses (Chan et al., 2001; Borzychowski et al.,

2005).

Natural killer (NK) cells have an important role in the early responses to viral

infections and have also been linked with failure of pregnancy. NK cells (identified

by the surface marker CD56) are the dominant type of maternal immune cell

populating the

uterine mucosa during formation of the placenta. Nowadays,

attention has been directed at their possible role in regulating the fetal supply line by

modulating the structural adaptation of the uterine spiral arteries. This is achieved

by invasion of the maternal decidua and adjacent myometrium by invasive fetal

trophoblast cells but trophoblast invasion is found to be defective in intrauterine

growth restriction, preeclampsia, and miscarriage (Moffett-King, 2004).


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NK cells play a fundamental role in the innate immune response through

their ability to secrete cytokines and kill target cells without prior sensitization.

These effector functions are central to NK cell anti-viral and anti-tumor abilities.

Due to their cytotoxic nature, it is vital that NK cells have the capacity to recognize

normal self-tissue and thus prevent their destruction. In addition to their role in host

defense, NK cells accumulate at the maternal-fetal interface and are thought to play

a critical role during pregnancy. The close proximity of uterine NK (uNK) cells to

fetal trophoblast cells of the placenta would seemingly lead to catastrophic

consequences, as the trophoblast cells are semi-allogeneic. A fundamental enigma

of pregnancy is that the fetal cells constitute an allograft but, in normal pregnancies,

they are in effect not perceived as foreign and are not rejected by the maternal

immune system (Riley and Yokoyama, 2008).

During normal conditions NK cells contribute to creating a favorable

environment for placentation, but at the same time they are equipped with cytotoxic

potential to fight intrauterine infections. Decidual NK cells are known to produce a

variety of cytokines; trophoblast cells express receptors for many of these

cytokines, indicating that they can potentially respond. In this way, decidual NK

cells have a significant influence on trophoblast behavior during implantation (Loke

and King, 2000). Decidual NK activity is regulated by a complex, mutually

interacting network of cytokines and hormones (Szekeres-Bartho, 2008).

Decidual NK cell responses are different in anembryonic pregnancies and in

recurrent spontaneous abortions than in normal pregnancies (Chao et al., 1995).

Attempts have also been made to compare the number of NK cells in the non-

pregnant endometrium of women with recurrent miscarriage or infertility with that

in normal controls and it has been found that the levels of NK cells developing in

presence of Th-1 type cytokines are also found in increased levels in peripheral

blood of non-pregnant women and pregnant women with a history of RPL as

compared to normal non-pregnant and pregnant women (Kwak et al., 1995).


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NKT cells are an unusual T cell subset capable of producing both Th1-like

and Th2-like cytokines. Unlike conventional alpha and beta T cells that recognize

peptides in the context of MHC molecule, NKT cells recognize glycolipids

presented by the MHC class I-like molecule, CD1d. Recent reports have

demonstrated that NKT cells and CD1d are present at the maternal-fetal interface.

Moreover, activation of NKT cells can have dramatic effects on pregnancy (Boyson

et al., 2008). During normal, intact pregnancy, peripheral blood NKr1 cells and

decidual NK3 cells increase, while the NKT cell populations decrease significantly

in miscarriage cases, suggesting an imbalance in NK1/NK2/NK3/NKr1 is correlated

with miscarriage (Saito et al., 2008).

Higuma-Myojo et al. (2005) supported the NK1/NK2/NK3/NKr1 hypothesis

by observing that the main populations of CD56bright NK cells and CD56dim NK

cells were IFN-gamma-producing NK1 type cells in peripheral blood of the non-

pregnant subjects. Populations of IL-10-producing NKr1 type cells in peripheral

blood CD56bright NK cells and CD56dim NK cells in early pregnant women were

significantly greater compared with those in non-pregnant women, and these cell

populations decreased in miscarriage cases. In the early pregnancy decidua, the

main populations of CD56bright NK cells and CD56dim NK cells were TGF-beta-

producing NK3 type cells, and NK1 type cells were rare. NK3 type cells in decidua

were significantly decreased in miscarriage cases compared with those in normal

pregnant subjects. IL-4-, IL-5- or IL-13-producing NK2 type cells were rare in

peripheral blood and decidua.

Whether produced by Helper T cells or natural killer cells, cytokines are

known to play a crucial role in success and failure of pregnancy. Moreover, it has

also been suggested that cytokine production of both helper T cells and natural

killer cells are dependent on each other and the interplay between them is

determinable for the pregnancy. Thus it becomes important to understand the

pathogenesis triggered by imbalance between pro-inflammatory and anti-


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inflammatory cytokines during pregnancy which results in various pregnancy

complications leading to fetal death.

1.3. Cytokines and Pregnancy

Cytokines are small, nonstructural proteins with molecular weights ranging

from 8 to 40,000d. Today the term cytokine is used as a generic name for a diverse

group of soluble proteins and peptides that act as humoral regulators at nano to

picomolar concentrations and which, either under normal or pathological

conditions, modulate the functional activities of individual cells and tissues. These

proteins also mediate interactions between cells directly and regulate processes

taking place in the extracellular environment. Many growth factors and cytokines

act as cellular survival factors by preventing programmed cell death or apoptosis.

Cytokines are immuno-modulatory proteins representing a group of proteins

and peptides that are used in organisms as signaling compounds allowing

communication between the cells. They are particularly important in both innate

and adaptive immune responses. Due to their central role in the immune system,

cytokines are involved in a variety of immunological, inflammatory and infectious

diseases. In addition they play a key role in neuro-immunological, neuro-

endocrinological, and neuro-regulatory processes. Cytokines are important positive

or negative regulators of the cell cycle, differentiation, migration, cell survival and

cell death, and cell transformation.

However, not all cytokine functions are limited to the immune system, as

they are involved in several developmental processes during embryogenesis. They

are important mediators involved in embryogenesis and organ development and

their activities in these processes may differ from those observed post nataly. It has

been reported that cytokines play an important role in success and failure of

pregnancy (Raghupathy, 1997).


19

Cytokine mediators can be transported quickly to remote areas of a

multicellular organism. They can address multiple target cells and can be degraded

quickly. Cytokines play a pivotal role in all sorts of cell-to-cell communication

processes although many of the mechanisms of their actions have not yet been

elucidated in full detail. A close examination of the physiological and pathological

effects of the regulated or deregulated expression of cytokines in complex

organisms has shown that these mediators are involved in virtually all general

systemic reactions of an organism including such important processes as the

regulation of immune responses, inflammatory processes, hematopoiesis and

wound healing.

It has been shown that a number of viral infectious agents exploit the

cytokine repertoire of organisms to evade immune responses of the host. Virus-

encoded factors appear to affect the activities of cytokines in at least four different

ways: by inhibiting the synthesis and release of cytokines from infected cells; by

interfering with the interaction between cytokines and their receptors; by inhibiting

signal transmission pathways of cytokines; and by synthesizing virus-encoded

cytokines that antagonize the effects of host cytokines mediating antiviral

processes. Bacteria and other micro-organisms also appear to produce substances

with activities resembling those of cytokines and which they utilize to subvert host

responses.

In many respects the biological activities of cytokines resemble those of

classical hormones produced in specialized glandular tissues. Some cytokines also

behave like classical hormones in that they act at a systemic level, affecting, for

example, biological phenomena such as inflammation, systemic inflammatory

response syndrome, and acute phase reaction, wound healing and the neuroimmune

network.

In general, cytokines act on a wider spectrum of target cells than hormones.

Perhaps the major feature distinguishing cytokines from mediators regarded

generally as hormones is the fact that, unlike hormones, cytokines are not produced


20

by specialized cells organized in specialized glands, i.e., there is not a single organ

source for these mediators. The fact that cytokines are secreted proteins also means

that the sites of their expression do not necessarily predict the sites at which they

exert their biological function.

Each cytokine has a matching cell-surface receptor. Subsequent cascades of

intracellular signaling then alter cell functions. This may include the up regulation

and/or down regulation of several genes and their transcription factors, resulting in

the production of other cytokines, an increase in the number of surface receptors for

other molecules, or the suppression of their own effect by feedback inhibition. The

cytokine receptors have come in attention of investigators than cytokines

themselves, partly because of their remarkable characteristics, and partly because a

deficiency of cytokine receptors has now been directly linked to certain debilitating

immunodeficiency states.

Based on three-dimensional structure, a classification of cytokine receptors

is given below. Such a classification, though seemingly cumbersome, provides

several unique perspectives for attractive pharmacotherapeutic targets.

• Immunoglobulin (Ig) superfamily, which is ubiquitously present throughout

several cells and tissues of the vertebrate body, and share structural

homology with immunoglobulins (antibodies), cell adhesion molecules, and

even some cytokines. Examples: IL-1 receptor types.

• Haemopoietic Growth Factor (type 1) family, whose members have certain

conserved motifs in their extra cellular amino-acid domain. The IL-2

receptor belongs to this chain, whose γ-chain (common to several other

cytokines) deficiency is directly responsible for the X-linked form of Severe

Combined Immunodeficiency (X-SCID).

• Interferon (type 2) family, whose members are receptors for IFN β and γ.

• Tumor necrosis factors (TNF) (type 3) family, whose members share a

cysteine-rich common extracellular binding domain, and includes several


21

other non-cytokine ligands like CD40, CD27 and CD30, besides the ligands

on which the family is named (TNF).

• Seven transmembrane helix family, the ubiquitous receptor type of the

animal kingdom. All G-protein coupled receptors (for hormones and

neurotransmitters) belong to this family. Chemokine receptors, two of which

act as binding proteins for HIV (CXCR4 and CCR5), also belong to this

family.

The effect of a particular cytokine on a given cell depends on the cytokine, its

extracellular abundance, the presence and abundance of the complementary receptor

on the cell surface, and downstream signals activated by receptor binding; the last

two factors can vary by cell type. Cytokines are characterized by considerable

"redundancy", in that many cytokines appear to share similar functions.

Generalization of functions is not possible with cytokines. Nonetheless, their

actions may be grouped as autocrine (if the cytokine acts on the same type of cell

that secretes it), paracrine (if the target is restricted to cells of a different type in the

immediate vicinity of a cytokine's secretion) and in some instances endocrine (if the

target is on distant cells).

Cytokines are produced by a variety of cells (both haemopoietic and non-

haemopoietic) and the same cytokine is even produced by different types of cells at

the same time in response to any foreign particle e.g. IFN family cytokines are

produced by Th-1 cells, NK cells and macrophages at the same time in response to

any viral infected cell. They have effects on both nearby cells and throughout the

organism and sometimes these effects are strongly dependent on the presence of

other chemicals and cytokines.

T cells are initially activated as Th0 cells, which produce IL-2, IL-4 and

IFN-γ. The nearby cytokine environment then influences differentiation into Th1 or

Th2 cells. IL-4 stimulates Th2 activity and suppresses Th1 activity, while IL-12

promotes Th1 activities. Th1 and Th2 cytokines are antagonistic in activity. Th1

cytokine IFN-γ inhibits proliferation of Th2 cells, while IFN-γ and IL-2 stimulate B


22

cells to secrete IgG2a and inhibit secretion of IgG1 and IgE. Th2 cytokine IL-10

inhibits Th1 secretion of IFN-γ and IL-2; it also suppresses Class II MHC

expression and production of bacterial killing molecules and inflammatory

cytokines by macrophages. IL-4 stimulates B cells to secrete IgE and IgG1. The

balance between Th1 and Th2 activities may steer the immune response in the

direction of cell-mediated or humoral immunity.

Helper T cells have two important functions: to stimulate cellular immunity

and inflammation and to stimulate B cells to produce antibody. Two functionally

distinct subsets of T cells secrete cytokines which promote these different activities.

Th1 cells produce IL-2, IFN-γ, and TNF-α, which activate Tc and macrophages to

stimulate cellular immunity and inflammation. Th1 cells also secrete IL-3 and GM-

CSF to stimulate the bone marrow to produce more leukocytes. Th2 cells secrete

IL-4, IL-5, IL-6, and IL-10, which stimulate antibody production by B cells.

Although various classifications for cytokines have been suggested on the basis of

their mode of action, structure and receptors, etc. but depending on their

inflammatory reactions, they are broadly categorized into pro-inflammatory and

anti-inflammatory cytokines which are produced by Th-1 and Th-2 cells,

respectively. Helper T cells are so called as they help in stimulating cellular

immunity and inflammation and also in stimulating B cells to produce various

antibodies. Two functionally distinct subsets of helper T cells secrete cytokines

which promote their different activities. Th-1 cells in general are associated with the

promotion of excessive inflammation and tissue injury. They produce pro-

inflammatory cytokines which activate TC and macrophages to stimulate cellular

immunity and inflammation. The Th-2 cells on the other hand are associated with

the antibody production and support the allergic reaction by producing anti-

inflammatory cytokines which act antagonistically to Th-1 type cytokines. The

effects of both pro-inflammatory and anti-inflammatory cytokines on the implanting

embryo and the outcome of pregnancy are depicted in Figure 1.1.


23

Helper T cells

Th-1 type cells Th-2 type cells

Pro-inflammatory cytokines Anti-inflammatory cytokines

(TNF-α, TNF-β, IFN-γ, IL-I, IL-2, etc) (IL-4, IL-6, IL-10, IL-12, etc)

Pregnancy failure Successful pregnancy

Figure 1.1: Schematic representation of the effects of Th-1 and Th-2 type cytokines

in pregnancy.

1.3.1. Anti-inflammatory (Th-2) Cytokines

Th-2 cells encourage antibody production and produce anti-inflammatory

cytokines that promote embryonic development and placentation. For instance, Il-4,

IL-6 and IL-10 are propitious to the success of pregnancy and deficiency of these

cytokines leads to poor placentation, subnormal growth and even sometimes fetal

death (Clark and Chaouat, 1989). These anti-inflammatory cytokines are also

known to control the action of Th-1 dependent cytokines as they act antagonistically

on Th-1 cells (Wegmann et al., 1993; Romagnani, 1994; Raghupathy, 1997) which

otherwise might attack fetus or the trophoblasts in general.

Cytokines such as IL-4, IL-10, IL-13and transforming growth factor (TGF)-b

suppress the production of IL-1, TNF, chemokines such as IL-8, and vascular

adhesion molecules. Therefore, a “balance” between the effects of pro-

inflammatory and anti-inflammatory cytokines is thought to determine the outcome


24

of disease, whether in the short term or long term. In fact, some studies have data

suggesting that susceptibility to disease is genetically determined by the balance or

expression of either pro-inflammatory or anti-inflammatory cytokines.

1.3.2. Pro-inflammatory (Th-1) Cytokines

Pro-inflammatory cytokines is a general term for those immunoregulatory

cytokines that favour inflammation. The major pro-inflammatory cytokines that are

responsible for early responses are IL1-alpha, IL1-beta, IL6 and TNF-alpha. Other

pro-inflammatory mediators include LIF, IFN-gamma, OSM, CNTF, TGF-beta,

GM-CSF, IL11, IL12, IL17, IL18, IL8 and a variety of other chemokines that

chemo attract inflammatory cells. These cytokines either act as endogenous

pyrogens (IL1, IL6, TNF-alpha), up-regulate the synthesis of secondary mediators

and other pro-inflammatory cytokines by both macrophages and mesenchymal cells

(including fibroblasts, epithelial and endothelial cells), stimulate the production of

acute phase proteins or attract inflammatory cells.

Th-1 cells are involved in cell-mediated inflammation and produce pro-

inflammatory cytokines which inhibit trophoblast growth and differentiation. Some

of the first studies on RPL associated abnormal immune reactivity in the context of

Th1 – Th2 paradigm demonstrated in vitro that trophoblast antigens activate

lymphocytes of RPL susceptible women to produce embryotoxic cytokines i.e.

TNF-α, IFN-γ and IL-2 (Yamada et al., 1994; Hill, 1995; Hill et al., 1995).

It is also well known that Th-1 type cytokines induce programmed cell death

(apoptosis), the effect of which could comprise the trophoblast barriers separating

the semiallogenic fetus from the mother’s immune system, leading to fetal rejection

or abortion. Th-1 type cytokines may also act by inducing the development of NK,

LAK and CTL cells that cause fetal death, as they are capable of killing

trophoblasts (Drake and Head, 1989).


25

The net effect of an inflammatory response is determined by the balance

between pro-inflammatory and anti-inflammatory cytokines. It should be noted that

the common and clear-cut classification of cytokines as either pro anti-

inflammatory or pro-inflammatory may be misleading. The type, duration, and also

the extent of cellular activities induced by one particular cytokine can be influenced

considerably by the nature of the target cells, the micro-environment of a cell,

depending, for example, on the growth and activation state of the cells, the type of

neighboring cells, cytokine concentrations, the presence of other cytokines, and

even on the temporal sequence of several cytokines acting on the same cell.

The concept that some cytokines function primarily to induce inflammation

and others suppress the inflammation is fundamental to cytokine biology and also to

clinical medicine. The concept is based on the genes coding for the synthesis of

small mediator molecules that are up-regulated during inflammation. For example,

genes that are pro-inflammatory are type II phospholipase (PL) A2, cyclooxygenase

(COX)-2 and inducible NO synthase. These genes code for enzymes that increase

the synthesis of platelet-activating factor and leukotrienes, prostanoids, and NO.

Another class of genes that are proinflammatory is chemokines, which are small

peptides (8,000d) that facilitate the passage of leukocytes from the circulation into

the tissues. The prototypical chemokine is the neutrophil chemoattractant IL-8. IL-8

also activates neutrophils to degranulate and cause tissue damage. IL-1 and TNF are

inducers of endothelial adhesion molecules, which are essential for the adhesion of

leukocytes to the endothelial surface prior to emigration into the tissues. Taken

together, pro-inflammatory cytokine mediated inflammation is a cascade of gene

products usually not produced in healthy persons. What triggers the expression of

these genes? Although inflammatory products such as endotoxins trigger it, the

cytokines IL-1 and TNF (and in some cases IFN-g) are particularly effective in

stimulating the expression of these genes. Moreover, IL-1 and TNF act

synergistically in this process. Whether induced by an infection, trauma, ischemia,

immune-activated T cells, or toxins, IL-1 and TNF initiate the cascade of


26

inflammatory mediators by targeting the endothelium (Figure 1.2). The general

information about the activities of three pro-inflammatory cytokines (known to be

related with RPL) considered in the present study are briefed in Table 1.1.

Table 1.1: Selected pro-inflammatory cytokines and their activities

Cytokine Producing Cell Target Cell Function

IFN-γ Th1 cells,

Tc cells, NK cells

Various Viral replication

Macrophages MHC expression

Activated B cells Ig class switch to IgG2a

Th2 cells Proliferation

Macrophages Pathogen elimination

TNF-α Macrophages, Mast

cells, NK cells Macrophages

CAM and cytokine

expression

Tumor cells Cell death

TNF-β Th1 and Tc cells Phagocytes

Phagocytosis, NO

production

Tumor cells Cell death


27

Figure 1.2: The inflammatory cascade triggered by IL-1 and TNF. [iNOS =

inducible NO synthase; PAF = platelet-activating factor]

Source: Dinarello (2000)

1.3.2.1. Tumor Necrosis Factor-alpha

Tumor Necrosis Factor (TNF) is a pleotropic pro-inflammatory cytokine

secreted by Th-1 (CD4+) cells. It is also produced by macrophages, monocytes,

neutrophils and Natural Killer cells following their stimulation by bacterial

lipopolysacharides. Stimulated peripheral neutrophilic granulocytes but also

unstimulated cells and as well as a number of transformed cell lines, astrocytes,

microglial cells, smooth muscle cells, and fibroblasts all secrete TNF. The synthesis

of TNF-alpha is induced by many different stimuli including interferons, IL2, GM-

CSF, bradykinin, Immune complexes, inhibitors of cyclooxygenase and platelet

activating factor (PAF). On the other hand, the production of TNF is inhibited by

IL6, TGF-beta, vitamin D3, prostaglandin E2, dexamethasone, CsA (Cyclosporin

A) and antagonists of PAF.

TNF is a family of cytokines that share a cysteine rich common extracellular

binding domain. These are also referred to as a group of cytokines that are capable


28

of causing apoptosis. The two molecular species of TNF are known as TNF-α

(Cachectin) and TNF-β (Lymphotoxin). They are also known as TNF superfamily

member 2 (TNFSF2) and TNF superfamily member 1 (TNFSF1) respectively and

are popularly named as TNF-α and LT-α respectively. Homology of TNF-alpha

with TNF-beta is approximately 30 %.

TNF was found originally in mouse serum after intravenous injection of

bacterial endotoxins into mice primed with viable Mycobacterium bovis, strain

Bacillus Calmette-Guerin (BCG). TNF was then shown to be present also in sera of

rats, rabbits and guinea pigs. TNF-containing serum from mice is cytotoxic or

cytostatic to a number of mouse and human transformed cell lines, but less or not

toxic to normal cells in vitro. It causes necrosis of transplantable tumors in mice.

Human TNF-alpha is a non-glycosylated protein of 17 kDa and a length of 157

amino acids. Murine TNF-alpha is N-glycosylated. The 17 kDa form of the factor is

produced by processing of a precursor protein of 233 amino acids. A TNF-alpha

converting enzyme has been shown to mediate this conversion.

TNF-alpha contains a single disulfide bond that can be destroyed without

altering the biological activity of the factor. Mutations Ala84 to Val and Val91 to

Ala reduce the cytotoxic activity of the factor almost completely. These sites are

involved in receptor binding. The deletion of 7 N-terminal amino acids and the

replacement of Pro8Ser9Asp10 by ArgLysArg yield a mutated factor with an

approximately 10-fold enhanced anti-tumor activity and increased receptor binding,

as demonstrated by the L-M cell assay, while at the same time reducing the toxicity.

Two receptors of 55-60 kDa and 75-80 kDa have been described for TNF-alpha.

The 55-60 kDa has been given the designation CD120a in the nomenclature of CD

antigens and is also referred to as TNFRSF1A [TNF receptor superfamily member

1A]. The gene encoding the production of TNF-alpha has a length of approximately

3.6 kb and contains four exons. The primary transcript has a length of 2762

nucleotides and encodes a precursor protein of 233 amino acids. The aminoterminal

78 amino acids function as a presequence.


29

The human TNF gene maps to chromosome 6p23-6q12 (Figure 1.3). It is

located between class 1 HLA region for HLA-B and the gene encoding complement

factor C. The gene encoding TNF-beta is approximately 1.2 kb downstream of the

TNF-alpha gene. However, both genes are regulated independently. The two genes

also lie close to each other on murine chromosome 17.

Figure 1.3: Location of TNF-alpha and TNF-beta genes on chromosome 6.

(Entrez Gene cytogenetic band: 6p21.3, Ensembl cytogenetic band: 6p21.33)

TNF-alpha shows a wide spectrum of biological activities. It causes cytolysis

and cytostasis of many tumor cell lines in vitro. Sensitive cells die within hours

after exposure to picomolar concentrations of the factor and this involves, at least in

part, mitochondria-derived second messenger molecules serving as common

mediators of TNF cytotoxic and gene-regulatory signaling pathways. The factor

induces hemorrhagic necrosis of transplanted tumors. Within hours after injection

TNF-alpha leads to the destruction of small blood vessels within malignant tumors.

The factor also enhances phagocytosis and cytotoxicity in neutrophilic granulocytes

and also modulates the expression of many other proteins, including fos, myc, IL1

and IL6. The 26 kDa form of TNF is found predominantly on monocytes and T-

cells after cell activation. It is also biologically active and mediates cell destruction

by direct cell-to-cell contacts.

TNF mediates part of the cell mediated immunity against obligate and

facultative bacteria and parasites. It confers protection against Listeria

monocytogenes infections, and anti-TNF antibodies weaken the ability of mice to


30

cope with these infections. TNF-alpha is a growth factor for normal human diploid

fibroblasts. It promotes the synthesis of collagenase and prostaglandin E2 in

fibroblasts. It may function also as an autocrine growth modulator for human

chronic lymphocytic leukemia cells in vivo and has been described to be an

autocrine growth modulator for neuroblastoma cells. The autocrine growth-

promoting activity is inhibited by IL4. In resting macrophages, TNF induces the

synthesis of IL1 and prostaglandin E2. It also stimulates phagocytosis and the

synthesis of superoxide dismutase in macrophages. TNF activates osteoclasts and

thus induces bone resorption. TNF-alpha inhibits the synthesis of lipoprotein lipase

and thus suppresses lipogenetic metabolism in adipocytes. In progenitors of

leukocytes and lymphocytes TNF stimulates the expression of class I and II HLA

and differentiation antigens, and the production of IL1, colony stimulating factors,

IFN-gamma, arachidonic acid metabolism. It also stimulates the biosynthesis of

collagenases in endothelial cells and synovial cells.

IL6 suppresses the synthesis of IL1 induced by bacterial endotoxins and

TNF, and the synthesis of TNF induced by endotoxins. The neurotransmitter

substance P induces the synthesis of TNF and IL1 in macrophages. IL1, like IL6,

stimulates the synthesis of ACTH (corticotropin) in the pituitary. Glucocorticoids

synthesized in response to ACTH in turn inhibit the synthesis of IL6, IL1 and TNF

in vivo, thus establishing a negative feedback loop between the immune system and

neuroendocrine functions. TNF-alpha enhances the proliferation of T-cells induced

by various stimuli in the absence of IL2. Some subpopulations of T-cells only

respond to IL2 in the presence of TNF-alpha. In The presence of IL2 TNF-alpha

promotes the proliferation and differentiation of B-cells.

The functional capacities of skin Langerhans cells are also influenced by

TNF-alpha. These cells are not capable of initiating primary immune responses such

as contact sensibilisation. They are converted into immunostimulatory dendritic

cells by GM-CSF and also IL1. These cells therefore are a reservoir for

immunologically immature lymphoid dendritic cells. The enhanced ability of


31

maturated Langerhans cells to process antigens is significantly reduced by TNF-

alpha. Although TNF-alpha is required also for normal immune responses the over

expression has severe pathological consequences. TNF-alpha is the major mediator

of cachexia observed in tumor patients. TNF is also responsible for some of the

severe effects during Gram-negative sepsis. TNF promotes the proliferation of

astroglial cells and microglial cells and therefore may be involved in pathological

processes such as astrogliosis and demyelinisation.

In vivo TNF-alpha in combination with IL1 is responsible for many

alterations of the endothelium. It inhibits anticoagulatory mechanisms and promotes

thrombotic processes and therefore plays an important role in pathological

processes such as venous thromboses, arteriosclerosis, vasculitis, and disseminated

intravasal coagulation. The expression of membrane thrombomodulin is decreased

by TNF-alpha. TNF-alpha is a potent chemoattractant for neutrophils and also

increases their adherence to the endothelium. The chemotactic properties of

Formyl-Met-Leu-Phe (fMLP) for neutrophils are enhanced by TNF-alpha. TNF-

alpha induces the synthesis of a number of chemoattractant cytokines, including IP-

10, JE, KC, in a cell-type and tissue-specific manner. Although TNF inhibits the

growth of endothelial cells in vitro it is a potent promoter of angiogenesis in vivo.

The angiogenic activity of TNF is significantly inhibited by IFN-gamma.

1.3.2.2. Tumor Necrosis Factor-beta

This factor is produced predominantly by mitogen-stimulated T-lymphocytes

and leukocytes. The factor is secreted also by fibroblasts, astrocytes, myeloma cells,

endothelial cells, epithelial cells and a number of transformed cell lines. The

synthesis of TNF-beta is stimulated by interferons and IL2. Some pre-B-cell lines

and Abelson murine leukemia virus-transformed pre-B-cell lines constitutively

produce TNF-beta.

TNF-beta is a protein of 171 amino acids N-glycosylated at position 62.

Some cell lines secrete different glycosylated forms of the factor that may differ


32

also in their biological activities. The protein does not contain disulfide bonds and

forms heteromers with LT-beta that anchors the complexes in the membrane.

Murine and human TNF-beta is highly homologous (74%). Recombinant human

proteins with deletions of 27 amino acids from the N-terminus appear to be

biologically active in several bioassays.

The gene encoding for TNF-beta has a length of approximately 3 kb and

contains four exons. It encodes a primary transcript of 2038 nucleotides yielding an

mRNA of 1.4 kb. The gene maps to human chromosome 6p23-6q12 approximately

1.2 kb apart from the TNF-alpha gene (Figure 1.3). The 5' region of the TNF-beta

promoter contains a poly (dA-dT)-rich sequence that binds the nonhistone protein

HMG-1 which is involved in the regulation of the constitutive expression of the

gene. TNF-beta binds to the same receptor as TNF-alpha.

TNF-beta acts on a plethora of different cells. This activity is not species-

specific. Human TNF-beta acts on murine cells but shows a slightly reduced

specific activity. In general, TNF-beta and TNF-alpha display similar spectra of

biological activities in vitro systems, although TNF-beta is often less potent or

displays apparent partial agonist activity. TNF-beta is cytolytic or cytostatic for

many tumor cells. In monocytes TNF-beta induces the terminal differentiation and

the synthesis of G-CSF. TNF-beta is a mitogen for B-lymphocytes. In neutrophils

TNF-beta induces the production of reactive oxygen species. It is also a

chemoattractant for these cells, increases phagocytosis, and also increases adhesion

to the endothelium. TNF-beta also induces the synthesis of GM-CSF, G-CSF, IL1,

collagenase, and prostaglandin E2 in fibroblasts. TNF-beta inhibits the growth of

osteoclasts and keratinocytes. Although TNF-beta binds to the same receptor as

TNF-alpha it is not involved in the establishment of an endotoxin shock. TNF-beta

promotes the proliferation of fibroblasts and is involved probably in processes of

wound healing in vivo. Hemorrhagic necrosis of tumors induced by TNF-beta in

vivo is probably the result of an inhibition of the growth of endothelial cells and the

activity of TNF-beta as an anti-angiogenesis factor.


33

Administration of TNF induces metabolic acidosis, decreases the partial

pressure of CO2, induces the synthesis of stress hormones such as epinephrine,

norepinephrine, and glucagon, and also alters glucose metabolism. It is well known

that TNF-α and TNF-β exert predominantly pro-inflammatory responses including

apoptosis (Hehlgams and Pfeffer, 2005). Due to their pro-inflammatory and pro-

apoptotic capacity, they are described to mediate several aspects of pregnancy

complications including pre-eclampsia (Anim-Nyame et al., 2003), miscarriage

(Babbage et al., 2001) and recurrent pregnancy loss (Rezaei and Dabbagh, 2002).

Several mechanisms were proposed for the pro-abortogenic effects of TNF-α and

TNF-β including trophoblast invasion and placentation (Kwak-Kim et al., 2005)

and induction of the expression of pro-apoptotic genes in human fetal membranes

(Garcia-Lloret et al., 2000), which in turn accelerates membrane degradation and

thus increases the susceptibility to premature rupture (Fortunato et al., 2001). They

are known to be cytotoxic to embryonic fibroblast like cells (Suffys et al., 1989) as

they interfere with the proliferation of human trophoblast lines (Haimovici et al.,

1991). It is also demonstrated using murine models that TNF-α terminates the

normal pregnancy when injected (Chaouat et al., 1990). It is also known to cause

fetal expulsion due to uterine contraction or may even cause necrosis of implanted

embryo or it could act by thrombosing the blood supply to conceptus (Raghupathy,

1997). It may also act by inducing the Natural Killer (NK), Lymphokine Activated

Killer (LAK) and Cytotoxic T Lymphocytes (CTL) cells that cause fetal death, as

they are capable of causing trophoblasts (Drake and Head, 1989; Raghupathy et al.,

2000). TNF-α is also reported to act along with the hormones and cause thromboses

in the placenta resulting in miscarriage and its production is enhanced at the onset

of labor and spontaneous abortion (Daher et al., 1999; Carp, 2006).


34

1.3.2.3. Interferon-gamma

Interferon-gamma was among the first cytokines identified (Wheelock,

1965). It is well characterized genetically, structurally, and functionally in many

species (Pestka et al., 2004; Schoenborn and Wilson, 2007). IFNG plays important

roles in diverse cellular processes, including activating innate and adaptive immune

responses, inhibiting cell proliferation, and inducing apoptosis (Boehm et al., 1997;

Stark et al., 1998). It is also crucial in immune responses against pathogens and

immunosurveillance of tumors (Boehm et al., 1997; Szabo et al., 2003; Dunn et al.,

2006).

By definition interferons are proteins that, at least in homologous cells, elicit

a virus-unspecific antiviral activity. This activity requires new synthesis of RNA

and proteins and is not observed in the presence of suitable RNA and protein

synthesis inhibitors. Interferons are mainly known for their antiviral activities

against a wide spectrum of viruses. Interferons are synthesized, for example, by

virus-infected cells and protect other, non-infected but virus-sensitive cells against

infection for some time. In addition interferons are also known to have protective

effects against some non-viral pathogens. Apart from their antiviral activities

interferons also possess antiproliferative and immunomodulating activities and

influence the metabolism, growth and differentiation of cells in many different ways

(Figure 1.4).

Interferons are also potent immunomodulators. They can promote or inhibit

the synthesis of antibodies by activated B-cells and also activate macrophages,

natural killer cells, and T-cells. Interferons mainly influence early unspecific

immune response processes mediated predominantly by monocytes/macrophages.

Among other things interferons increase antigen and receptor expression in effector

cells, induce the expression of new genes, inhibit the expression of some genes, and

also prolong phases of the cell cycle. Interferons also influence differentiation and

developmental processes, which is exemplified by their effects on the maturation of


35

immature muscle cells, the induction of globin genes, the methylation of tRNA, and

the expression of carcinoembryonic antigen on tumor cells.

Figure 1.4: Schematic representation of various activities of Interferon

Interferons also possess direct antiproliferative activities and are cytostatic or

cytotoxic for a number of different tumor cell types. These activities are partly due

to complex interactions with other growth factors and their receptors the expression

of which may be stimulated or inhibited by interferons. Many growth factors are

capable also of inducing the synthesis of interferons. Hormone-like activities of

interferons are observed in cells of the central nervous and the neuroendocrine

system. The three main human interferons are known as IFN-alpha, IFN-beta and

IFN-gamma. IFN-alpha and IFN-beta as well as IFN-delta, IFN-omega, and IFN-

Interferon

Binding to specific

menberane receptors Gene activation

Antiviral Activity

Inhibition of viral DNA replication

Antiproliferative Activity

Alterations of cell membranes

Alteration of cytoskeleton

Stimulation of cell differentiation

Module of growth factor expression inhibition / induction of

oncogene expression

Reversion of malignant cell phenotypes

Immunomodulatory Activity

Induction of cytokine expression

Activation of macrophages

Activation of lymphocytes

Upregulation of HLA Class I and II expression

Modulation of expression of tumor associated antigens


36

tau are also called Type 1 interferon. IFN-gamma has been designated Type 2

interferon. Type 3 interferon is a collective term referring to IL28A, IL28B, and

IL29.

IFN-gamma does not display significant homology with the other two

interferons, IFN-alpha and IFN-beta. Murine and human IFN-gamma show

approximately 40 sequence homology at the protein level. Interferon (IFN)-g is

another example of the pleiotropic nature of cytokines. Like IFN-a and IFN-b, IFN-

g possesses antiviral activity. IFN-g is also an activator of the pathway that leads to

cytotoxic T cells. However, IFN-g is considered a pro-inflammatory cytokine

because it augments TNF activity and induces nitric oxide (NO). IFN-gamma is

produced mainly by T-cells and natural killer cells activated by antigens, mitogens,

or alloantigens. It is produced by lymphocytes expressing the surface antigens CD4

and CD8. The synthesis of IFN-gamma is induced, among other things, by IL2,

bFGF, and EGF. B-cells also produce IFN-gamma, and constitutive synthesis has

been observed in many established human B-cell lines. On the other hand, the

synthesis of IFN-gamma is inhibited by 1-alpha,25-Dihydroxy vitamin D3,

dexamethasone and CsA (Cyclosporin A).

IFN-gamma is synthesized as a precursor protein of 166 amino acids

including a secretory signal sequence of 23 amino acids. Two molecular forms of

the biologically active protein of 20 and 25 kDa have been described. Both of them

are glycosylated at position 25. The 25 kDa form is also glycosylated at position 97.

The observed differences of natural IFN-gamma with respect to molecular mass and

charge are due to variable glycosylation patterns. 40-60 kDa forms observed under

non-denaturing conditions are dimers and tetramers of IFN-gamma. Recombinant

IFN-gamma isolated from Escherichia coli is also biologically active and

glycosylation therefore is not required for biological activity. IFN-gamma contains

two cysteine residues that are not involved in disulfide bonding.

At least six different variants of naturally occurring IFN-gamma have been

described. They differ from each other by variable lengths of the carboxyterminal


37

ends. The biological activities of these variants do not differ from recombinant IFN-

gamma obtained from Escherichia coli. It has been proposed that at least some of

these variants are the result of proteolytic cleavage by exopeptidases and hence

constitute purification artifacts. In contrast to IFN-alpha and IFN-beta IFN-gamma

is labile at pH 2. IFN-gamma can exist in a form associated with the extracellular

matrix and may therefore exert juxtacrine growth control.

The human gene encoding for IFN-gamma has a length of approximately 6

kb. It contains four exons and maps to chromosome 12q24.1 (Figure 1.5).

Figure 1.5: Location of IFN-gamma gene on chromosome 12

(Entrez Gene cytogenetic band: 12q14, Ensembl cytogenetic band: 12q15)

IFN-gamma has antiviral and antiparasitic activities and also inhibits the

proliferation of a number of normal and transformed cells. IFN-gamma synergises

with TNF-alpha and TNF-beta in inhibiting the proliferation of various cell types.

The growth inhibitory activities of IFN-gamma are more pronounced than those of

the other interferons. However, the main biological activity of IFN-gamma appears

to be immunomodulatory in contrast to the other interferons that are mainly

antiviral.

In T-helper cells IL2 induces the synthesis of IFN-gamma and other

cytokines. IFN-gamma acts synergistically with IL1 and IL2 and appears to be

required for the expression of IL2 receptors on the cell surface of T-lymphocytes.

Blocking of the IL2 receptor by specific antibodies also inhibits the synthesis of

IFN-gamma. IFN-gamma thus influences cell mediated mechanisms of cytotoxicity.


38

IFN-gamma is a modulator of T-cell growth and functional differentiation. It is a

growth-promoting factor for T-lymphocytes and potentiates the response of these

cells to mitogens or growth factors. The human promyelocytic leukemia cell line

HL-60 can be induced to differentiate by a number of stimuli. IFN-gamma, but not

other interferons, specifically induces differentiation of these cells into monocytes.

IFN-gamma inhibits the growth of B-cells induced by IL4. IFN-gamma and Anti-Ig

co-stimulate the proliferation of human B-cells but not of murine B-cells. IFN-

gamma also inhibits the production of IgG1 and IgE elicited by IL4 in B-cells

stimulated by bacterial lipopolysaccharides. IFN-gamma regulates the expression of

MHC class 2 genes and is the only interferon that stimulates the expression of these

proteins.

IFN-gamma also stimulates the expression of Ia antigens on the cell surface,

the expression of CD4 in T-helper cells, and the expression of high-affinity

receptors for IgG (like CD16, CD32, CD64) in myeloid cell lines, neutrophils, and

monocytes. In monocytes and macrophages IFN-gamma induces the secretion of

TNF-alpha and the transcription of genes encoding G-CSF and M-CSF. In

macrophages IFN-gamma stimulates the release of reactive oxygen species. IFN-

gamma is involved also in processes of bone growth and inhibits bone resorption

probably by partial inhibition of the formation of osteoclasts.

IFN-gamma inhibits the proliferation of smooth muscle cells of the arterial

intima in vitro and in vivo and therefore probably functions as an endogenous

inhibitor for vascular overreactions such as stenosis following injuries of arteries.

IFN-gamma inhibits the proliferation of endothelial cells and the synthesis of

collagens by myofibroblasts. It thus functions as an inhibitor of capillary growth

mediated by myofibroblasts and fibroblast growth factors and PDGF. IFN-gamma

specifically induces the transcription of a number of genes. These genes contain

regulatory DNA sequences within their promoter regions (ISRE; Interferon-

stimulated response element) that function as binding sites for a number of


39

transcription factors. Some of these genes are expressed also in response to other

interferons.

IFN-γ is also known to inhibit embryonic and fetal development as well as

the proliferation of human trophoblast lines like TNF-alpha (Haimovici et al., 1991)

as both these cytokines are cytotoxic to embryonic fibroblast like cells (Suffys et

al., 1989). It is also reported that IL-2, TNF-α and IFN-γ together terminate normal

pregnancy when injected (Chaouat et al., 1990). Also, IFN-γ inhibits secretion of

GM-CSF from uterine epithelium necessary for successful pregnancy (Robertson et

al., 1994).

IFNG has been widely assessed as a potential mediator of many

complications of human pregnancy as well. In normal pregnancies, semi-allogeneic

trophoblast cells are not subject to transplant rejection reactions by maternal

lymphocytes. This may be due in part to intrinsic regulatory mechanisms that

prevent IFNG-induced expression of MHC molecules, a pathway of immune-

evasion known for tumors and cells infected by certain viruses. However,

gestational complications that include fetal loss have been linked to elevation in

IFNG (Murphy et al., 2009). Shi et al. (2007) have highlighted the significance of

the level of IFN-γ during pregnancy by concluding that they found no change of

total type 1 and type 2 lymphocytes in human early pregnancy, however, IFN-γ was

decreased in NK cells and NKT cells.

1.4. Th-1 Bias in Pregnancy Failure

The late Tom Wegmann first proposed that fetal survival depends on a bias

of maternal immune responses towards T-helper Th2 immunity and the inhibition of

cytotoxic Th1 responses (Wegmann et al., 1993).

Peripheral blood mononuclear cells (PBMCs) from a significant number of

women with a history of RPL showed a greater cell proliferation and produced

soluble factors that were toxic to mouse embryos and human trophoblast lines when

stimulated in vitro with trophoblast antigen extracts (Yamada et al., 1994; Hill et


40

al., 1995). Out of 244 women with unexplained RPL, 160 were shown to have

PBMCs that responded in vitro to trophoblast antigens by producing embryotoxic

activity and high levels of pro-inflammatory cytokines but a very low level of anti-

inflammatory cytokines. Conversely, women who were not prone to RPL responded

without production of Th-1 type cytokines but had IL-10 (Th-2 type cytokine)

activity (Hill et al., 1995).

It has been investigated by enzyme linked immuno sorbent assay (ELISA)

testing that there is increased production of pro-inflammatory cytokines (Th-1 type)

and reduced production of anti-inflammatory cytokines (Th-2 type) in women with

recurrent pregnancy losses suggesting Th-1 bias in pregnancy failure and Th-2 bias

in successful pregnancy (Wegmann et al., 1993; Tangri and Raghupathy, 1993;

Tangri et al., 1994; Yamada et al., 1994; Hill, 1995; Hill et al., 1995) suggested that

these may be etiological factors in recurrent miscarriages (Mueller-Eckharat et al.,

1994; Jenkins et al., 2000; Raghupathy et al., 2000).

It was also shown by dot-blot and northern hybridization techniques that the

expression of TNF-α, IFN-γ and IL-2 is significantly greater in placentas of

abortion prone pregnancies compared with those of normal pregnancies (Tangri and

Raghupathy, 1993). Tangri and his colleagues using Elisa and bioassay suggested a

significantly greater production of TNF-α, IFN-γ and IL-2 in mixed lymphocyte

placental reaction (MLPR) supernatants in abortion prone mating combinations

compared to normal combinations (Tangri et al., 1994) where IFN-γ was produced

at 55-fold greater concentration, TNF-α at 10-fold greater concentration and IL-2 at

25-fold greater concentration. These studies suggest that a Th-2 bias is necessarily

maintained in case of normal pregnancy to act antagonistically to Th-1 cytokines

whereas reproductive failure is characterized by Th-1 bias.


41

1.5. Th-2 to Th-1 Switch

Various causes have been suggested for the possible mechanisms that shift

Th-2 to Th-1 dominant environment in pregnancy failure. Hill and colleagues

suggest that women with RPL may have a fundamental aberration in the regulation

of immune responses that skews the pattern from Th-2 type to Th-1 type cytokines

in reproductive failure (Hill, 1995; Hill et al., 1995).

The Th-2 to Th-1 shift in pregnancies may be due to one or more factors

(Figure 1.6) such as the deficiency of some putative immune-modulatory molecules

like PIBF, placental factors, IL-10 and TGF-β2. It is also likely that a balance

between IL-12 (favoring Th-1 response) and IL-4 (favoring Th-2 response)

determines the eventual outcome of the Th-1 – Th-2 dichotomy during an immune

response (Trinchieri, 1993).

Infection during pregnancy, particularly by intracellular parasites, may well

be an important factor that drives the response in a certain direction. Th-1 type cells

induced by the infection may traverse the fetal interface or may produce cytokines

that affect the trophoblasts (Krishnan et al., 1996). It is also assumed that a previous

abortion due to some other cause may prime the mother for subsequent Th-1 biased

responses (Hill, 1995). Infections with agents such as Toxoplasma Gondii and CMV

that lead to predominantly cellular immune responses and the production of pro-

inflammatory cytokines have been associated with recurrent miscarriages (Stray-

Pederson and Stray-Pederson, 1984; Lim et al., 1996). Such infections may prime

the mother to produce pro-inflammatory responses in subsequent pregnancy (Hill,

1995).

Although, various causes have been reported for the possible mechanism that

shifts Th-2 to Th-1 dominant environment in pregnancy loss, the production of pro-

inflammatory and anti-inflammatory cytokines is also found to be partly under

genetic control (Messer et al., 1991; Wilson et al., 1997).


42

1.6. Cytokine Gene Polymorphisms and RPL

Genetic polymorphisms associated with high and low production of a

number of cytokines including TNF-α, TNF-β, IFN-γ, IL-1, IL-2, IL-6 and IL-10

have been found (Messer et al., 1991; Wilson et al., 1992; Wilson et al., 1997;

Turner et al., 1997; Pravica et al., 1999; Bidwell et al., 2001; Daher et al., 2003). In

view of the cytokine gene polymorphisms known to cause elevated levels of pro-

inflammatory cytokines and thus RPL, it was suspected that women carrying these

polymorphisms might be genetically predisposed to developing habitual abortions.

Few studies have been performed to investigate the association between recurrent

Figure 1.6: Relationship between Th2 and Th1 type reactivity with successful

pregnancy and pregnancy failure, respectively.

Source: Raghupathy (1997)


43

pregnancy loss and the above described cytokine gene polymorphisms. The studies

concerning recurrent pregnancy loss and the polymorphisms known to cause greater

expression of TNF-α TNF-β and IFN-γ pro-inflammatory cytokine genes i.e. TNF-α

(-308 G/A; rs1800629), TNF-β (+252 G/A; rs909253) and IFN-γ (+874 A/T;

rs2436051) are discussed below.

1.6.1. Single Nucleotide Polymorphisms

Numerous single nucleotide polymorphisms (SNPs) have been reported in

TNF-α gene, but the one present in the promoter region, especially G/A

polymorphism at -308 position is known to cause an increased production of TNF-

α cytokine (Wilson et al., 1997). Also, a A/G SNP in 1st intron region of TNF-β

gene at +252 position correlates with the polymorphism in codon 26; wherein,

TNF-β*A allele (mutated) is associated with a reduced level of TNF-β production

and TNF-β*G allele is strongly associated with increased production of TNF-β

cytokine (Messer et al., 1991; Zammiti et al., 2008, 2009). Again a large number of

polymorphisms are reported in IFN- γ gene but specifically A/T polymorphism at

+874 position in the intronic region is known to cause an overexpression of the gene

and thus resulting in an increased production of IFN- γ cytokine (Pravica et al.,

1999). Few studies have been performed in the recent past regarding the association

of the above three mentioned polymorphisms with various inflammatory diseases

like renal disorders, leishmaniasis, arthritis and recurrent pregnancy losses.

The published data between 2001 and 2007 regarding recurrent spontaneous

abortions (RSA) and cytokine gene polymorphisms were reviewed by Choi and

Kwak-Kim (2008) to provide comprehensive understanding and a direction for the

future investigations. Either allele and/or genotype frequencies of the following

polymorphisms were reported to be significantly different between women with

RSA and controls: IFN-gamma +874A-->T, TA (p = 0.01), AA (P = 0.04); IL-6, -

634C-->G CG/GG (p = 0.026); IL-10, -592C-->A CC (p = 0.016); IL-1B -511C (p


44

= 0.035), -31T (p = 0.029); IL-1RA, IL1RN*2 (p = 0.002), and IL1RN*3 (p =

0.002). They concluded that multiple cytokine polymorphisms were reported to be

associated with RSA. However, a majority of the studies reviewed were not

confirmed or refuted by other investigators. Inconsistent study results might be

related to the following reasons.

(i) the production of these cytokines is partly under genetic control and other

factors affect cytokine levels,

(ii) ethnic background, environmental factors and selection criteria for study

populations are different, and

(iii) the possibilities exist that multiple cytokine gene polymorphisms or other

genes in linkage disequilibrium may play a role in RSA.

On the other hand, to assess and synthesize the available data from association

studies of inflammatory cytokine polymorphisms with RPL, a systematic review

and random effect meta-analysis of genetic association studies was performed by

Bombell and McGuire (2008). Sixteen reports of genetic association studies of

cytokine polymorphisms with RPL were identified and this analysis conducted on

their findings did not identify any significant association with tumor necrosis factor

(-308A, or -238A), interferon-gamma (+874T), interleukin (IL)-1beta (-511T), IL-6

(-174G) or IL-10 (-1082A or -819T or -592A). Significant associations were found

with IL-1B (-31T) (two studies: pooled odds ratio (OR) 2.12 (95% confidence

interval (CI) 1.04 to 4.33)) and IL-6 (-634G) (one study: OR 0.22 (95% CI 0.09 to

0.57)). The authors concluded that the available data are not consistent with more

than modest associations between these candidate cytokine polymorphisms and

RPL. An overview of studies conducted to reveal association, if any, between the

three selected cytokine gene polymorphisms viz., TNF-α (-308 G/A; rs1800629),

TNF-β (+252 A/G; rs909253) and IFN-γ (+874 A/T; rs2436051) and recurrent

pregnancy loss in various populations of the world is presented in Table 1.2.


45

Table 1.2: Outline of association studies reported using pro-inflammatory cytokine

gene polymorphisms and RPL

Authors Subjects Markers Result

Babbage et

al (2001)

Cases: 43

Caucasian women

of UK

Controls:73

Caucasian ward-

staff of UK

TNF-α (-308)

IFN-γ (+874)

No association found

Baxter et al

(2001)

Cases: 76 British

Caucasian couples

Controls: 69 British

Caucasian couples

TNF-α (-238),

TNF-α (-308),

TNF-β (Codon 26),

TNF-β (+252)


Reid et al

(2001)

Caucasian women

of UK

TNF-α (-308) Increased incidence

of TNF-α*G allele

among cases

Daher et al

(2003)

Cases: 48 Brazilian

Caucasian women

Controls: 108

healthy Brazilian

Caucasian

individuals (both

females and males)

TNF-α (-308)

IFN-γ (+874)

Trend towards

increased

frequencies of TNF-

α (-308) A/A and

A/G genotypes in

cases

Positive association

between IFN-γ

(+874) and RPL


46

Pietrowski

et al (2004)

Cases: 168 white

Caucasian women

Controls: 212 white

caucasian women

TNF-α (-308)


Prigoshin et

al (2004)

Cases: 41 Argentine

women

Controls: 54

Argentine women

TNF-α (-308)

IFN-γ (+874)


w.r.t. TNF-α (-308)


between IFN-γ

(+874) and RPL

Kamali-

Sarvestani

et al (2005)

Cases: 139 women

of Iran

Control: 143

women of Iran

TNF-α (-308)

TNF-β (+252)


Zamitti et al

(2008)

Cases: 350 women

of Tunisia

Controls: 200

women of Tunisia

TNF-α (-238)

TNF-α (-308)

TNF-β (+252)


w.r.t. TNF-α (-238)

& TNF-β (+252)

Zamitti et al

(2009)

Cases: 372 women

of Tunisia

Controls: 274

women of Tunisia

TNF-α (-238)

TNF-α (-308)

TNF-β (+252)


w.r.t. TNF-α (-238)

& TNF-β (+252)

Babbage et al. (2001) performed a case-control study in which 43 Caucasian

women (aged 21 – 45 years) suffering from RPL attending a particular hospital in

UK and 73 Caucasian ward staff women (ages 30 – 58 years) as controls were

included. Both the groups (cases and controls) were screened for TNF-α (-308

G/A) and IFN-γ (+874 A/T) along with some other markers and they reported no

association between RPL and either of the two molecular markers. The authors


47

concluded that either the genetic factors are not a major determinant of cytokine

production during pregnancy or the observed differences in cytokine production by

peripheral lymphocytes do not accurately indicate what is occurring at the local

materno-foetal interface during pregnancy. However, Reid et al. (2001) assessed the

carriage of rarer alleles of TNF-α*2 and IL-1β*2 among women with recurrent

miscarriages and observed an increased incidence in the carriage of TNF-α*2 more

pronounced in the women with two or more sequential miscarriages as compared to

the normal women. Daher et al. (2003) screened 48 Brazilian Caucasian women

with unexplained RPL and 108 healthy Brazilian Caucasian individuals (82 females

and 26 males) for TNF-α (-308 G/A) and IFN-γ (+874 A/T) along with IL-10 (-

1082 A/T) and also performed a meta-analysis including their own data and all the

previous studies mentioned above. The results showed statistically higher

frequencies of IFN-γ genotype TT (+874) i.e. p=0.04 and its positive association

with RPL (OR=1.92) as well as a trend towards increased frequencies of A/A and

A/G (-308) TNF-α genotypes when compared to general population (p=0.18 and

OR=1.31). They also concluded that these polymorphisms exert a detrimental effect

on pregnancy development.

On the other hand, Pietrowski et al. (2004) performed an association based

case-control study on Caucasian women, including 168 unexplained RPL cases in

the study group and 212 in the control group concerning two TNF-α (-308 and -

863) sites and concluded that these polymorphisms and resting blood TNF-α levels

do not correlate with the propensity to RPL in Caucasian women.

Prigoshin et al. (2004) studied TNF-α (-308) and IFN-γ (+874)

polymorphisms along with other pro-inflammatory and anti-inflammatory cytokine

gene polymorphisms in Caucasian Argentine women (41 with RPL and 54 controls)

and showed a significant association between RPL versus controls concerning IFN-

γ (+874 A/T) where TA genotype was found to be more in the patient group (65%

versus 35.8%, p=0.01). They supported the concept of IFN-γ (+874 A/T) being

involved in pathogenesis of unexplained RPL. However, no association was found


48

between RPL versus controls concerning the TNF-α (-308) polymorphism. Kamali-

Sarvestani et al. (2005) performed a similar case-control study on Iranian women

with reference to TNF-α (-308), TNF-β (+252) and IFN-γ (+874) polymorphisms

along with other Th-1 and Th-2 cytokine gene polymorphisms in which 139 women

with unexplained RPL in the study group and 143 women in the control group were

investigated and it was concluded that there is no association between the selected

molecular markers and the manifestation of RPL.

Zammiti et al. (2008, 2009) conducted a study among women of Tunisia

with respect to TNF-α (-308 G/A), TNF-α (-238 G/A) and TNF-β (+252 G/A)

polymorphisms and recurrent miscarriages. They divided the patients into various

stages of pregnancy loss and reported a positive association between exclusively

early idiopathic recurrent miscarriages and TNF-α (-238) GA and AA and TNF-β

(+252) AG genotypes but not with TNF-α (-308 G/A) polymorphism.

1.6.2. TNF-αααα and TNF-ββββ Haplotypes

Besides the individual association of genetic polymorphisms with the

manifestation of a disease, an attempt is being made nowadays to understand the

impact of SNP’s when present together using haplotype analysis. Such an approach

is likely to project a clearer picture about the role of various candidate genes in the

expression and degree of manifestation of a disorder. Keeping the above view in

mind, a couple of studies have been conducted with respect to TNF-alpha and TNF-

beta haplotypes and RPL. Baxter et al. (2001) screened British Caucasian couples

(76 cases and 69 controls) for TNF-α (-238 G/A), TNF-α (-308 G/A), TNF-β

(Codon 26) and TNF-β (+252 G/A) polymorphisms and found four major

haplotypes among their subjects similar to that reported by Fanning et al. (1997) i.e.

GGthrA, GGasnG, GAasnG and AGthrA, but found no association between any of

these haplotypes and the prevalence of recurrent miscarriages. This study

hypothesized that elevated maternal and fetal levels of TNF and TNF (-308)


49

polymorphism are associated with premature membrane rupture and preterm

delivery (Roberts et al., 1999; Ferriman et al., 2000) but found no association

between RPL and variant allele of TNF-α. Whereas, Zammiti et al. (2008, 2009)

while conducting a study among women of Tunisia with respect to TNF-α (-308

G/A), TNF-α (-238 G/A) and TNF-β (+252 G/A) polymorphisms, reported two

susceptible haplotypes i.e. TNF-α -308 A / TNF-α -238 G / TNF-β +252 G and

TNF-α -308 G / TNF-α -238 A / TNF-β +252 G, which were found to play a

vulnerable role in idiopathic recurrent miscarriages in regression analysis. They

even identified a protective haplotype TNF-α -308 A / TNF-α -238 G / TNF-β

+252 A in their sample.

Different reasons have been given by various researchers to explain the

absence of association between RPL and pro-inflammatory cytokine gene

polymorphisms known to cause elevated levels of respective cytokines in blood.

Babage et al. (2001) proposed that the cytokine production by human peripheral

blood lymphocytes does not mirror the response of immune cells at the materno-

fetal interface because of the differences in antigenic environment and type of

immune cells in the circulation and the placenta (Vince and Johnson, 1995; Vives et

al., 1999). Further, the innate rather than acquired immune response is supposed to

be playing a critical role in determining outcome of pregnancy by some authors

(Sacks et al., 1999). Another explanation given by them was that the previously

observed differences in the cytokine production could be due to other factors like

infections occurring during pregnancy which may prime the mother to produce pro-

inflammatory responses. However, the cases recruited in the present study were

ruled out for the presence of any infectious agent by appropriate examination.

Pietrowski et al. (2004) justified their findings by the dynamic expression of TNF-

α reflected at the protein level corresponding to various environmental or analogous

factors. For instance it was shown by Raghupathy et al. (1999, 2000) that the


50

antigen-stimulated peripheral blood mononuclear cells shift to Th2 bias in normal

and towards a Th1 bias in RPL women.

1.7. The Present Study

Figure 1.7 illustrates the gene-protein-phenotype relationship in recurrent

pregnancy loss. Although elevated levels of TNF-α and TNF-β are known to be

associated with pregnancy complications including recurrent pregnancy loss (b),

and TNF-α and TNF-β single nucleotide polymorphisms resulting in increased

production of these cytokines is also well established (a), still a direct correlation

between these cytokine gene polymorphisms and incidence of recurrent pregnancy

loss is controversial (c) (see Figure 1.7).

Figure 1.7: Gene – Protein – Phenotype relationship in RPL

However, the association between the candidate markers and the

manifestation of the disease is known to be population specific. It is believed that

the occurrence of a mutation, its propogation and its association with the causation

and expression of a disease is also population, geography and environment specific

(Walia et al., 2008). The extent of the manifestation of the disease vary in different

ethnic groups as they are largely influenced by the mating patterns, surrounding

genetic environment and life style and other environmental factors which are

population specific. Further, the frequency of these genetic polymorphisms reported

TNF-α (308)

TNF-β (252) IFN- γ (874)

TNF-α, TNF-β & IFN-γ

Pregnancy Loss

c

b a


51

in one particular population cannot be generalized for any other population group,

especially in the Indian context as people of India comprise of numerous

endogamous caste and tribal groups that maintain their relatively intact gene pools.

Moreover, the selected molecular markers i.e. TNF-α (-308) TNF-β and IFN-γ

(+874) are the candidate genes for the recurrent pregnancy loss and act as one of the

possible causes of the disease along with the other genes in the region which are

also influenced by the environmental causes.

Although few such studies have been conducted on some populations of the

world, no data have been reported on any Indian populations concerning the

association of cytokine gene polymorphisms and recurrent pregnancy loss. Further

it has always been a challenge to collect representable number of unexplained

recurrent pregnancy loss cases and even controls for drawing any meaningful

interpretation. Keeping this in mind, the present study was planned to observe the

genetic status of habitual abortion in North India and to understand the immuno-

molecular etiology of otherwise unexplained recurrent pregnancy loss in the Indian

population.

1.7.1. Rationale

Since the occurrence of a mutation, its propagation and its association with

the causation and expression of a disease is population, geography and environment

specific, the findings of the association studies (via candidate gene approach)

performed in different parts of the world cannot be generalized for the Indian

population as well. Thus, there is a need for population specific screening of various

candidate markers to observe their relative frequency in each population. Therefore,

the present study was planned in order to replicate and validate the suspected

molecular markers related to RPL (identified through candidate gene approach) in

the North Indian population. In addition, since the detection of these

polymorphisms in a woman also helps in treating and managing the pregnancy of a

carrier via immunotherapy, the suggested population specific screening approach


52

would also help in establishing population / individual specific pharmaco-genomic

and counseling approach.

1.7.2. Aim

The present study has been undertaken to reveal an association, if any,

between the pro-inflammatory cytokine gene polymorphisms known to be

responsible for their elevated expression, and otherwise unexplained recurrent

pregnancy loss in population of Delhi, North India.

1.7.3. Hypothesis

Null Hypothesis: The pro-inflammatory cytokine gene polymorphisms viz., TNF-α

(-308 G/A), TNF-β (+252 A/G) and IFN-γ (+874 A/T) are not associated with RPL

and the higher frequency among the cases may be because of chance factor.

Alternative Hypothesis: The selected pro-inflammatory cytokine gene

polymorphisms are significantly associated with RPL and the higher frequency

among the cases may not be because of chance factor.

1.7.4. Objectives

In order to test the above mentioned null hypothesis and fulfill the aim of the

present disease association study the following objectives were postulated.

1. To find out the frequency distribution of TNF-α (-308 G/A), TNF-β (+252

A/G) and IFN-γ (+874 A/T) polymorphisms among the women with

recurrent pregnancy loss and the age matched controls in population of

Delhi.

2. To estimate the extent of association between the three selected candidate

markers and RPL in population of Delhi.


53

3. To understand the dynamics of these polymorphisms with respect to

individual genes and also all of them taken together through haplotype

analysis.

4. To find out the presence of linkage disequilibrium in TNF-α and TNF-β

polymorphisms in the population of Delhi.

5. To estimate the extent of association between TNF-α and TNF-β haplotypes

and RPL in population of Delhi.

1.7.5. Significance

As no study is reported on any Indian population regarding the role of

cytokine gene polymorphisms in recurrent pregnancy loss, the present investigation

was planned and it aims to reflect upon the risk of immunologically mediated

pregnancy losses in the population of Delhi in North India. If an association is

found between selected molecular markers and recurrent pregnancy loss, this could

explain the cause of otherwise unexplained recurrent pregnancy losses. This study is

also likely to stimulate similar studies in various other ethnic groups inhabiting

other regions of India, which could bring out community specific associations,

further leading to population / individual specific pharmacological approach in

recurrent pregnancy loss.

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