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.