Cytokine imbalance at materno-embryonic interface and RPL
The endometrium originates during early stages of development from
uro-genital ridge and develops in uterus [185]. It is composed of
two main layers- the basal and functional layers. The basal layer is
located adjacent to myometrial layer and regenerates the functional
layer which is shed during menstruation. On the other hand the
functional layer that forms two-third of the thickness of endometrium
performs the role of secretion, proliferation and degeneration of tissue
[186]. The endometrium contains stromal and epithelial cells
supplied with a specific vasculature and this uterine lining attains
maturity at the age of puberty as well as orchestrates menstrual cycle
despite the fact that it provides an immuno-protected location for
embryonic allograft implantation. However, the tissues including
prostate and breasts that are proliferative and responsive to hormones
make the endometrial tissue pathology-prone [185]. The environment
of endometrium has a very important role in the embryo implantation and
the placental development during early pregnancy. In case of normal
pregnancy, the partly-allogenic embryo survives due to the stimulation
of maternal immune tolerance via Treg cells along with anti-inflammatory
Th2 profile [51]. A number of studies reported the imbalance of
immune cells (Table 6) and cytokine expression (Table 7 & 8) among
human females that suffer from RPL and implantation failure [187].
Imbalance in immunological reactions and deregulation in the activity of
subsets of T cells may produce reproductive failure such as miscarriage
[106]. Higher Th17 cell percentage and lower Treg cell number has
been reported in the decidua of women with idiopathic RPL compared to
that of normal controls suggesting the association of alterations in
Th17 and Treg cell ratio at materno-embryonic interface with the
pathogenesis of idiopathic RPL [220]. The type 1 cytokines including
IFNγ, IL17, IL1β and TNFα exert proinflammatory effects while the type 2
cytokine that produce anti-inflammatory effects include IL4, IL10 and
IL1ra [221]. The equilibrium between the proinflammatory and
anti-inflammatory is essential for implantation [222]. Prior to the
blastocyst arrival, the stromal cells of endometrium release
proinflammatory cytokines such as TNFα and IL1β to trigger inflammatory
activity [16] that can attain optimum efficiency at implantation
window which is the period of an immune and inflammatory reaction
[223]. The early identification of immune and inflammatory reaction
initiators such as TNFα and IL1β may act as predicators of implantation
[223]. TNFα has been found linked with the process of inflammation
associated with implantation, placentation, and pregnancy outcome
[224]. The uterine endometrium is always in an active state
[161] The successful implantation requires the endometrium to
undergo vital alterations [225]. The endometrium undergoes
decidualization so that the uterus is prepared for the implantation of
embryo and gestation. During the event of decidualization, the
epithelium, stroma and blood vessels of the endometrium are changed into
the specific tissue termed decidua [117-119]. The outcome of
imperfect decidual development during the early months of gestation
might be infertility or complications of pregnancy, for instance,
preeclampsia, recurrent pregnancy loss and pre-term birth in later
months of gestation [9, 99, 180, 181].
The essential requirement for successful implantation of embryo is
inflammatory reaction associated with augmented expression of various
inflammatory cytokines and chemokines in endometrial cells as well as
blastocyst [226]. This early inflammation related with the
implantation of embryo is a physiologic response that initiates during
decidualization [226]. Kosova et al. reported that RPL patients show
allelic difference and variation in the expression of several cells of
the immune system such as white blood cells, lymphocytes as well as the
activation of T cells [227]. The maintenance of equilibrium between
the promotion and inhibition of immunity has been proposed as an
essential aspect for the development of fetus [161, 228]. The immune
homeostasis and tolerance occur at decidua which is comprised of many
maternally derived cells including decidual natural killer (dNK) cells,
macrophages, T cells, dendritic cells, B cells and NKT cells
(Table 9 ) [60, 229]. The dNK cells that originate from uNK
cells primarily appear in the endometrial tissue during the luteal phase
of menstrual cycle [60, 229]. These cells are mainly involved in the
promotion of trophoblastic invasion and remodeling of blood vessels in
order to tremendously increase the process of placental perfusion that
seems to be under the regulation of chemokine and cytokine expression
[229,230]. The main leukocytes that are present in non-pregnant
stromal tissue include lymphocytes and macrophages. Towards the end of
the luteal phase of endometrial cycle, the number of pheno-typically
unique uNK cells and macrophages increases. During early phase of
pregnancy the number of uNK cells further increases to such an extent
that these cells comprise 70% of cells derived from bone marrow in the
first trimester decidua of pregnancy [37]. The augmented
infiltration of uNK and Foxp3+ Treg cells into the
endometrium and diminished expression of PGRMC1 has been found
associated with RM [231]. The hormonally responsive epithelial and
stromal cell layers of endometrium show the expression of cytokine and
steroid receptors as well as generate cytokines [13, 232]. The
alteration in the array of decidual cytokines may well lead to
spontaneous pregnancy loss [12]. The reduced IL11 expression in the
endometrium of RPL patients suggests the potential role of this very
cytokine in preventing miscarriages [233]. T cell cytokines may
directly influence the growth and function of trophoblast cells as they
express receptors for different factors including IFNγ, TNFα, TGFβ, IL6,
LIF, M-CSF or they may stimulate macrophages that could assault the
trophoblast. The decidual macrophages are stimulated by IFNγ and produce
NO and TNFα that may cause pregnancy loss. The functions of macrophages
and Th1 type cells may be inhibited by IL4 and IL10 cytokine that
prevents the rejection of feto-allograft [234-236]. M-CSF
(macrophage colony stimulating factor) acts on the trophoblastic cells
and stimulates their proliferation [237]. It also triggers the
process of differentiation among cyto-trophoblastic cells that as a
result grow into syncytium [238]. LIF (Leukemia inhibitory factor)
has a vital role in embryo implantation and development [239]. LIF
is usually produced by epithelial cells of endometrium and NK cells but
evidences show that this factor is additionally synthesized by certain
cells which are alike to Th2 type cells. T cells of decidua have been
reported to produce deficient LIF in RPL patients. Since cytokines have
the property to act locally as a result the measurement of levels of
cytokines produced by T cells at the maternal-embryonic interface show
significant results as compared to the measurement of their levels in
peripheral blood. Therefore, the examination of decidual T cells seems
to present an excellent technique for the study of pregnancy loss. The
diminished generation of decidual T cell cytokines especially IL4 and
IL10 has been reported among idiopathic RPL women as compared to women
with a history of normal pregnancy [82]. However, a few studies have
shown association between successful pregnancy and the T cell factors
such as IL4, IL10 and M-CSF produced at the materno-embryonic interface
[240]. IL10 expression at materno-embryonic interface has been
reported in numerous studies [24, 241,242] and this cytokine
inhibits the formation and role of proinflammatory cytokines such as
IL1, IL12, IFNγ etc [243]. During normal pregnancy, the
up-regulation of IL10 protein and mRNA has been found in gestational
tissues [244]. IL10 as a strong factor stimulates the production of
tolerogenic dendritic cells [245] which are key participants in
inducing materno-immune tolerance to embryonic allo-antigens [122]
and thus may promote prolific pregnancy.
T regulatory lymphocytes that produce as well as develop under the
influence of IL10 also play a significant role in pregnancy success via
inducing materno-immune tolerance to embryonic allo-antigens [51].
Binding of IL10 to IL10 receptor (IL10R) activates the IL10/JAK1/STAT3
cascade with subsequent STAT3 phosphorylation that results in STAT3
homo-dimer (STAT3/STAT3) production. The translocation of this
homo-dimer into nucleus stimulates the expression of the target gene
that plays a part in successful pregnancy induction [246-248]. The
phosphorylated STAT3 (pSTAT3) that significantly increases in decidual
Treg cells of idiopathic RPL patients inhibits Treg cell proliferation
via down-regulating STAT5 and Foxp3 expression and increases the
proportion of responder T cells. In addition, pSTAT3 decreases the IL10
and TGFβ1 secretion from Treg cells. The over-expression of IL6 and IL23
stimulates the phosphorylation of STAT3 in Treg cells. STAT3
hyper-phosphorylation impairs Treg cell cytokine secretion, suppression
and proliferation, while the inhibition of STAT3 phosphorylation
reinstates these Treg cell functions. pSTAT3 alters the balance of
Treg/Th17 at the materno-embryonic interface [249]. Reduced
production of IL10 together with the augmented synthesis of inflammatory
molecules might be a milieu that promotes preterm birth or loss of early
gestation [250]. However, the treatment of abortion-prone CBA/DBA
mice with recombinant IL10 significantly abrogates the rate of
spontaneous embryonic loss [251]. The human endometrial cells
including epithelial, stromal and lymphoid express TNFα along with its
mRNA [252, 253]. The endometrial TNFα level has been found dynamic
during menstrual cycle but showing gradual rise at late luteal phase
[254]. TNFα along with IFNγ hamper the growth of embryo and the
production of trophoblastic cell lines [255] due to their cytotoxic
effect on fibroblast like embryonic cells [256]. Higher level of
TNFα and IFNγ have been reported in circulating blood of women that
undergo successive pregnancy loss than those that carry gestation
successfully to full term [257, 258]. The equilibrium between IL12
and IL4 favours the response of Th1 and Th2 respectively and ultimately
decides the influence of Th1/Th2 dichotomy throughout an immune reaction
[259]. Furthermore, it is well understood that an earlier pregnancy
loss as a result of several other etiology might induce successive Th1
biased immune responses in mother [260].
The PGD2 (Postaglandin D2) synthesized by the placenta may work as a
chemo attractant of Th2 type cells to the maternal embryonic interface
through the expression of typical Th2 receptor molecule CRTH2
(Chemo-attractant receptor homologous molecule expressed on Th2 type
cells) [261]. The cells that express CRTH2+ are decreased in number
at the materno-embryonic interface among those females who experience
RPL in comparison to females that undergo elective termination of
pregnancy [262]. During pregnancy, infection mainly by intracellular
parasites might be a significant factor responsible for driving the
immune response along a specific course. Strongly Th1 predominant
reactions in opposition to infectious microbes compromise gestation; for
instance, the infection due to Leishmania major leads to resorption
followed by simultaneous rise in placental IFNγ level [263]. TNFα in
coordination with hormones causes placental thrombosis in pregnancy and
shows augmented synthesis with the inception of labor and pregnancy loss
[264]. TNFα exhibits pleiotropic property and is chiefly produced by
NK cells, mononuclear phagocytes, lymphocytes and antigen-stimulated T
cells [265]. TNFα and LTα (lymphotoxin-α) are known to produce
proabortogenic effects including the invasion of trophoblasts and
placentation [266] stimulation of pro-apoptotic gene expression in
human embryonic membranes [267] that in turn hastens the degradation
of membranes and their early rupture [268]. It also assists indirect
spontaneous pregnancy loss via stimulating NK cells or macrophages
[269]. The NK cells are stimulated by IFNγ produced by Th1 type of
cells and their activation might be injurious to murine conceptus
[270] showing the obvious relationship of NK cells and IFNγ in
pregnancy collapse. The cytokines produced by Th2 type of cells effect
NK cells in a number of ways that include inhibiting NK cell binding and
vascular endothelium cytotoxicity [271], hampering the production of
NK cells [272] and biasing of NK cell cytokine manufacture toward a
Th2 phenotype [273, 274]. Generally, uNK cells promote invasion of
trophoblasts, protect the embryo from maternal immune attack, and
enhances angiogenesis [275, 276] as suggested by their close
proximity with invading trophoblasts during early stage of pregnancy
[275, 277] and production of a broad spectrum of cytokines such as
TNFα, TGFβ, IFNγ, IL2, GM-CSF, LIF, CXCL10, CSF-1, and CXL12 as well as
angiogenic mediators such as VEGF (vascular endothelial growth factor)
and ANG2 (angiopoietin-2) on activation [278]. Additionally, uNK/dNK
cells express specific receptors for HLAs (human leukocyte antigens)
including KIR (killer immunoglobulin receptor), CD94/NKG2A, ILT2 which
are exclusively expressed in trophoblasts [279, 280]. This
interactive collaborative between uNK/dNK and trophoblast specific HLAs
is implicit as a vital potential factor implicated in the prevention of
embryonic rejection by maternal immune system [281- 284]. Moreover,
IL15 stimulated uNK cells are believed to play a key role in the
maintenance of homeostasis by selectively targeting and cleaning of
senescent decidual cells via granule exocytosis [285]. However,
there are reports that show abnormally higher number of NK cells among
females who have experienced idiopathic RPL [286]. A recent study
also reported considerably higher pNK cells in pregnant women with RPL
history patients as compared to normal pregnant females [287]. Most
recently it has been shown that pNK cytotoxicity is significantly
reduced in RPL patients than in fertile women [288]. Another most
recent study depicted results contrary to previous study and reported an
elevation in NK cytotoxic activity in RPL women compared to fertile
women [289]. Therefore, it seems that pregnancy-promoting functions
of NK cells are dysregulated in RPL patients.
Non-immune cells such as, the trophoblasts of the placenta furthermore
promote the dominance of Th2 type cytokines during pregnancy [290].
Trophoblast, decidua and amnion all act as contributing factors to the
Th2 type cytokine atmosphere via synthesizing IL13 [291], IL10
[292], IL4 and IL6 [293, 294]. Therefore, the production of any
of these cytokines in abnormal proportions in women affected with
idiopathic RPL might cause aberrant growth and role of placenta and
subsequently leads to spontaneous pregnancy loss [7]. The cytokine
environment in the endometrium can be regulated by T lymphocytes. Th2
type cytokines have been additionally reported in peri-implantation
endometrium of human beings [295] and at the murine
materno-embryonic interface [25], where they are thought to perform
a crucial part in the uterine cytokine network of pregnancy [296]
More TNF-α has been reported in the deciduas of RPL women who had
miscarried [297] however, less in their trophoblast [298]. In
human female endometrium, the augmented or suppressed Th1 immunity has
been observed during habitual implantation failure [299]. This
augmented Th1 type immunity might diminish the number or role of Treg
cells and the decline in the Treg cell number perhaps induces
implantation failure, resulting in idiopathic infertility [7].
Jasper et al. reported that primary idiopathic infertility is linked
with the diminished expression of Foxp3 mRNA in the endometrial tissue
[300]. Foxp3 mRNA and protein levels have been found decreased in
RPL affected women. The selective Foxp3 expression in human trophoblasts
suggests the association of Foxp3 expression with the trophoblast
proliferation and invasion behavior. The reduced expression of Foxp3 in
RPL trophoblasts may provide better insight in the RPL pathology
[301]. Treg cells take part in the protection of the embryo via
down-regulating inflammatory reactions. Treg cells have the potential to
inhibit the production of cytokines both in CD4+T and CD8+ cells,
cytotoxicity action of NK cells, as well as maturation and role of
dendritic cells, which suppresses the activation of local inflammation
[9, 302-304]. The lack of Treg cell mediated modulation may result
in loss of pregnancy [9]. The decidua of women with RPL has been
reported to show the significant expression of IL23, IL17 and retinoid
orphan receptor C [89]. The impaired dynamics and expression of
proinflammatory and implantation factor, such as IL33 [305] and
prokineticin-1 [306] form the molecular basis of decidual
dysfunction in RPL. IL33 regulates the factors essential for decidual
receptivity during the implantation window, whereas prokineticin-1
participates in angiogenesis and regulation of decidualization and
implantation processes [119]. Endometrium has been found low
receptive in idiopathic RPL women [307]. Impaired differentiation of
stromal cells has been reported in RPL [306]. Usually
proinflammatory cytokines including TNFα, IL1, IL6 [70] and IFN𝛾
produced by the recruited NK cells and Mo/Mph [308] are activated
locally in the decidua nearly the occasion of embryo implantation. These
cytokines affect the decidualization of DSCs (dendritic stromal cells)
and as a result determine the crosstalk between immune and non-immune
cells in the differentiating decidual environment. Later on,
anti-inflammatory factors enrich the decidual niche and support the
maternal immune tolerance towards the embryo [119]. The pregnancy
outcome shows a close association with the activities of different
biological factors especially cytokines (Table 10) whose imbalance at
materno-embryonic interface may cause reproductive failure. Differences
have been reported among endometrial immune cell populations of RPL
women before conception and become aggravated in women who subsequently
continue to miscarry [7]. This suggests that the immune network of
RPL females in fact, might be compromised before the establishment of
pregnancy. This nature of immune negotiation is still an arena of active
research.