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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
Chapter I: Introduction and Review of Literature
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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.
Chapter I: Introduction and Review of Literature
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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
Chapter I: Introduction and Review of Literature
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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
Chapter I: Introduction and Review of Literature
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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
Chapter I: Introduction and Review of Literature
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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
Chapter I: Introduction and Review of Literature
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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).
Chapter I: Introduction and Review of Literature
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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
Chapter I: Introduction and Review of Literature
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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).
Chapter I: Introduction and Review of Literature
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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
Chapter I: Introduction and Review of Literature
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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.
Chapter I: Introduction and Review of Literature
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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).
Chapter I: Introduction and Review of Literature
14
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).
Chapter I: Introduction and Review of Literature
15
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).
Chapter I: Introduction and Review of Literature
16
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).
Chapter I: Introduction and Review of Literature
17
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-
Chapter I: Introduction and Review of Literature
18
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).
Chapter I: Introduction and Review of Literature
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
Chapter I: Introduction and Review of Literature
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
Chapter I: Introduction and Review of Literature
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
Chapter I: Introduction and Review of Literature
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.
Chapter I: Introduction and Review of Literature
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
Chapter I: Introduction and Review of Literature
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).
Chapter I: Introduction and Review of Literature
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
Chapter I: Introduction and Review of Literature
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
Chapter I: Introduction and Review of Literature
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
Chapter I: Introduction and Review of Literature
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.
Chapter I: Introduction and Review of Literature
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
Chapter I: Introduction and Review of Literature
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
Chapter I: Introduction and Review of Literature
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
Chapter I: Introduction and Review of Literature
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.
Chapter I: Introduction and Review of Literature
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).
Chapter I: Introduction and Review of Literature
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
Chapter I: Introduction and Review of Literature
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
Chapter I: Introduction and Review of Literature
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
Chapter I: Introduction and Review of Literature
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.
Chapter I: Introduction and Review of Literature
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
Chapter I: Introduction and Review of Literature
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
Chapter I: Introduction and Review of Literature
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.
Chapter I: Introduction and Review of Literature
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).
Chapter I: Introduction and Review of Literature
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)
Chapter I: Introduction and Review of Literature
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
Chapter I: Introduction and Review of Literature
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.
Chapter I: Introduction and Review of Literature
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)
No association found
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
Chapter I: Introduction and Review of Literature
46
Pietrowski
et al (2004)
Cases: 168 white
Caucasian women
Controls: 212 white
caucasian women
TNF-α (-308)
No association found
Prigoshin et
al (2004)
Cases: 41 Argentine
women
Controls: 54
Argentine women
TNF-α (-308)
IFN-γ (+874)
No association found
w.r.t. TNF-α (-308)
Positive association
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)
No association found
Zamitti et al
(2008)
Cases: 350 women
of Tunisia
Controls: 200
women of Tunisia
TNF-α (-238)
TNF-α (-308)
TNF-β (+252)
Positive association
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)
Positive association
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
Chapter I: Introduction and Review of Literature
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
Chapter I: Introduction and Review of Literature
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)
Chapter I: Introduction and Review of Literature
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
Chapter I: Introduction and Review of Literature
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
Chapter I: Introduction and Review of Literature
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
Chapter I: Introduction and Review of Literature
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.
Chapter I: Introduction and Review of Literature
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.