Fig. 14.1
Defective receptivity, implantation, and/or decidualization can lead to infertility. Deferred implantation past the window of receptivity can lead to misguided embryo placement and implantation, resulting in placenta previa, ectopic placentation (placenta accreta) or placental insufficiency resulting in intrauterine growth restriction (IUGR) and/or preeclampsia. Implantation beyond the normal window can also give rise to spontaneous abortion, miscarriage and recurrent pregnancy loss, leading to infertility. Premature decidual senescence can lead to preterm birth and fetal death , whereas shallow trophoblast invasion into maternal decidua and/or blood vessels can lead to preeclampsia. [Reprinted Cha, J., X. Sun, and S.K. Dey, Mechanisms of implantation: strategies for successful pregnancy. Nature medicine, 2012. 18(12): p. 1754–1767. With permission Nature Publishing Group]
Decidualization is a postovulatory process, driven mainly by the progesterone secretion from the corpus luteum , in which the endometrium is prepared for embryo implantation and pregnancy. Human endometrial stroma cells differentiate from fibroblast-like into secretory and receptive decidual endometrial stromal cells. It happens every cycle, during the mid-secretory phase, irrespective of pregnancy [20]. A novel hypothesis named “menstrual preconditioning” might have the answer for why the decidualization process happens each cycle, ending in most cases in menstrual bleeding and shedding of the decidua [21].
In “menstrual preconditioning” the repetitive, short exposures to harmful stimuli to a degree below the threshold for tissue injury will provide some degree of protection from subsequent injury—as will happen when the trophoblast deeply invades into the endometrium during pregnancy. It appears that decidualization grants valuable characteristics to the endometrium, including embryo defense against environmental and oxidative stress, regulation of trophoblast invasion, and protection of the uterus from aggressive invasion by the embryo.
Only after the decidualization process do the endometrium stroma cells have the ability to act as biosensors of the embryo quality and react to low quality embryos by shutting down production of key implantation mediators and immunomodulators such as IL-1b, -6, -10, -17, -18, eotaxin, and heparin-binding EGF-like growth factor, thus preventing it from implanting [22]. This process is considered to be “embryo selection” in humans.
What Happens When There Is Impaired Embryo Selection?
Understanding decidualization’s important role led to the idea that aberrations during the decidualization and implantation might give rise to pregnancy with embryos who would otherwise be rejected by the decidua , resulting in early embryo loss. For example, a study of 221 women attempting to conceive demonstrated dramatic risk, increasing on a daily basis, of early miscarriage if pregnancy was established beyond the normal “implantation window” [23]. Correspondingly, recent observations succeeded in identifying several differences between women who suffer from RPL and no-RPL women: Women suffering from RPL express lower levels of mucin 1, an anti-adhesion molecule that contributes to the barrier function of luminal epithelium [24–26]. Another research showed different levels of two maker genes : higher levels of prok1, which encodes prok1, a cytokine that promotes endometrium receptivity, and lower levels of prolactin, a prototypic marker of decidualizing endometrial cells [27].
It was also shown that decidualized endometrial stromal cells from women who suffer from RPL fail to discriminate between low and high quality embryos when it comes to migrating towards the embryo at the site of implantation, unlike non-RPL women (as seen in Fig. 14.2) [28].
Fig. 14.2
The migration zone after adding a high-quality, low-quality or no embryo. The migratory response of decidualized H-EnSCs from normally fertile (a–c) and RM women (d–f) was analyzed in absence of a human embryo (a, d), in presence of a high-quality embryo (b, e) or a low-quality embryo (c, f). Phase contrast pictures were taken 18 h after creating the migration zone. The dotted line represents the front of the migration zone directly after its creation. As a reference for the position of the embryo, the bottom of the plate was marked. The arrows indicate the position of the embryo. All pictures were taken with 25× magnification. [Reprinted from Weimar, C.H., et al., Endometrial stromal cells of women with recurrent miscarriage fail to discriminate between high-and low-quality human embryos. PLoS One, 2012. 7(7): p. e41424. With permission from PLoS One]
The lack of embryo natural selection might provide an explanation for chromosomal and nonchromosomal pregnancy failures.
Superfertility and RPL
One could describe clinic al pregnancies as “the tip of the iceberg”—representing only 40 % of all conceptions, when 30 % of all conceptions are lost before implantation and an additional 30 % are lost before 6 weeks gestation [21].
The odds for a fertile couple to achieve pregnancy is ~20 % during one menstrual cycle, defined as the Monthly Fecundity Rate (MFR), odds that are considered to be very low compared to other mammalian species [29].
Using the MFR, a mathematical model predicts that 74, 93, and 100 % of normally fertile couples will achieve pregnancy in 6, 12, and 24 months.
Subfertility and superfertility have been defined by MFRs of 5 % or less, and 60 % or more, respectively [29]. According to the Tietze Model, it has been estimated that 79 % of the population is fertile, 18 % subfertile or infertile, and 3 % superfertile [30].
This relative inefficiency is sometimes considered to represent a strategy dealing with embryo chromosomal abnormalities, which is accepted as the most common cause for miscarriage, including recurrent miscarriage [31].
Recent observations have shown that as much as 40 % of the women with RPL can be defined as superfertile, with time-to-pregnancy (TTP) shorter than 3 months [27]. Rapid conceptions are associated with risk of early pregnancy loss even in low-risk populations, which can partly be explained by the lack of natural embryo selection.
In summary, we might learn from the low efficacy of human reproduction the role of cyclic decidualization of the endometrium and its importance.
Teklenburg et al. [22] coined the term “window of natural embryo selection,” as it reflects the functional role of decidualizing stromal cells in assessing the implanting embryo’s quality, and moreover the decidualizing stromal cells end the window of endometrial receptivity and enable the mother to dispose of embryos who are not “high quality.”
The most significant conclusion might be preventing early pregnancy complications and failures by targeting the endometrial decidu al response prior to pregnancy or immediately after implantation.
Heparin Use in Recurrent Pregnancy Loss
Heparin is a very widely used injectable anticoagulant and, according to the World Health Organization’s List of Essential Medicines, it is one of the most important medications needed in a basic health system [32].
It was originally isolated from canine liver cells, hence its name (hepar is Greek for “liver”) in 1916, but it wasn’t until the mid-30s that heparin was manufactured in a safe, nontoxic form, easily available, thus making it a popular anticoagulant. In the human body, heparin is stored exclusively in the granules of subsets of mast cells [33]. Although it is used principally in medicine for anticoagulation, its true physiological role in the body is still uncertain, since blood anticoagulation is achieved mostly by heparan sulfate proteoglycans derived from endothelial cells [34].
Almost 100 years after heparin’s discovery, nowadays best known for its first described anticoagulant ability, it is being researched for its other abilities as well. Heparin is useful in RPL in very specific indications but there is an option that some other less-known characteristics of the drug have impact on RPL outcome that come from other medical fields. Heparin is believed to possess many biological activities that include the ability to modulate embryonic development, neurite outgrowth, tissue homeostasis, wound healing, metastasis, cell differentiation, cell proliferation, and inflammation [33].
In this chapter we’ll see which of those attributes is pertinent in the RPL clinic through heparin’s effect on different molecules in in vitro studies and in vivo experiments.
Heparin’s Effect on Different Molecules
Although our knowledge about heparin’s anticoagulant properties has developed in the last century, discoveries about heparin’s anti-inflammatory features are comparatively new (see Chap. 2 for elaborated information). Heparin affects several molecules, including cytokines, growth factors, adhesion molecules, cytotoxic peptides, and tissue-destructive enzymes, many of which are crucially involved in the inflammatory process. Each of these proteins might be essential in better understanding the inflammatory component in RPL, and might help to decrease this phenomenon.
During the process of implantation heparin, and heparin-derived molecules, affect through expression of adhesion molecules, matrix-degrading enzymes, and trophoblast phenotype and apoptosis [35].
LMWH was shown to increase matrix metalloproteinase (MMPs) concentrations and activity and reduce their tissue inhibitor (TIMP) in a dose-dependent manner. Two of those MMPs, a group of matrix-degrading enzymes, were found to be necessary for the embryo’s ability to degrade the basement membrane of the uterine epithelium and to invade the uterine stroma [36]. Additionally, It was shown that LMWH induces an increased decidual expression and secretion of heparin-binding EGF-like growth factor (HB-EGF) and reduced TNF-alpha-induced apoptosis [37]. LMWH induces activation of a DNA-binding transcription factor that, once activated, enhances HB-EGF expression [38]; heparin has the ability to activate the EGF receptor in primary villous trophoblasts [39].
During the first trimester, fetal and placental development takes place in a low O2 tension environment, which is important to prevent complications related to exposure with normal concentrations of oxygen, such as preeclampsia, IUGR, and miscarriage [40]. HB-EGF has an important role in preventing hypoxic-induced apoptosis in the early stages of placentation. Heparin was also found to prevent apoptosis in human trophoblasts triggered by pathological and other stimuli ((IFN)-γ, (TNF)-α, thrombin, staurosporine) and activated survival signal transduction pathways [39].
More information on the effects of heparin on different molecules is elaborated in Table 14.1.
Table 14.1
Proteins involved in the inflammatory response that are bound by heparin and related molecules
Examples of heparin-binding inflammatory mediators | (Patho)physiological significance | Examples | Comments |
|---|---|---|---|
Adhesion molecules | Cell transport | CD11b/CD18 [132] (MAC1) | – |
– | – | L-selectin [133] | |
Chemokines [137] | Inflammatory cell recruitment and activation; viral infection | RANTES, IL-8, MIP1, MCP1, eotaxin | – |
– | – | PF4 | A minimum length of GAG chain is required for binding of PF4, a property that is exploited in improving the side-effect profile of heparin as an anticoagulant [138] |
Growth factors [137] | Tissue repair and repai ring; angiogenesis | PDGF, VEGF, TGF-β | – |
– | – | FGF2 | Binding affinity is related to both length and composition (L-iduronic acid content) of the GAG chain [139]. FGF2 signal transduction requires binding of heparin-sulfate (or heparin) to both FGF2 and its receptor. 2-O-sulphation is essential for the former and 6-O-sulphation for the latter. Therefore, selectively 6-O-desulphated heparin competitively inhibits FGF2-induced angiogenesis [140] |
Enzymes [137] | Digestion of tissue/ECM structural components | Heparanase69 MMPs | – |
– | – | Elastase cathepsin | As well as directly binding elastase and cathepsin G, heparin is thought to modulate the activity of these enzymes through potentiation of their natural inhibitor, SLPI. Heparin binds SLPI with greater affinity than less-sulphated GAGs, although within heparin, undersulphated chains bind with the highest affinity [141] |
Cytotoxic mediators [137] | Destr uction of parasites; tissue damage | ECP MBP | – |
In Vitro Models Succeeded in Showing Some of Heparin’s Beneficial Effects
1.
Dextran sulfate sodium (DSS)-induced colitis symptoms in a mouse model were found to be relieved by LMWH. It was found to inhibit the expression of IL-1b and IL-10 in the intestinal mucosa of DSS-induced colitis thus downregulating the expression of inflammatory cytokine production [41].
2.
When given after ischemia, heparin and N-acetylheparin were found to reduce the extent of myocardial injury associated with regional ischemia and reperfusion in the canine heart, in a mechanism of cytoprotection that is unrelated to alterations in the coagulation cascade and may involve inhibition of complement activation in response to tissue injury [42].
3.
The effects of single administrations of aerosolized heparin, LMWH, were examined on antigen-induced airway hyperresponsiveness and leukocyte accumulation in neonatal immunized rabbits, and were found to significantly inhibit the development of airway hyperresponsiveness if given prior to antigen challenge [43].
In Vivo Experiments
Several small clinical trials have shown heparin can be helpful in several inflammatory diseases thanks to its anti-inflammatory qualities.
1.
More than 20 years ago, it was discovered that inhaled heparin prevents exercise-induced asthma when given to the patients 45 min before their exercise. The researchers’ hypothesis was that this non-anticoagulant ability was more likely related to heparin’s modulation on mediator release rather than to an effect on smooth muscle [44].
2.
In a reported study, patients with ulcerative colitis unresponsive to high dose corticosteroid therapy were treated with heparin. Twelve out of 16 patients showed marked improvement in the disease activity [45], although meta-analyses of this and similar trials have concluded that there is currently insufficient evidence to support the use of heparin for the treatment of active ulcerative colitis [46].
3.
In a different study enoxaparin was shown to be of benefit when given to COPD patients and a significant increase in forced expiratory volume in 1 s (FEV1) over baseline was observed. A possible mechanism is heparin’s ability to inhibit the activity of neutrophil-derived proteases such as elastase and cathepsin G, or other neutrophil activities including degranulation, the respiratory burst, and processes involved in neutrophil trafficking into tissues. Given that COPD is an airways inflammatory disease with a predominance of neutrophils, the researchers hypothesized that heparin may have some therapeutic benefit in patients with COPD [47]. More diseases affected by non-anticoagulant attributes of heparin are elaborated in Table 14.2.
Table 14.2
Conditions (other than thrombosis ) in which heparin has been reported to confer benefit
Condition level of evidence | Level of evidence |
|---|---|
Acute respiratory distress syndrome/acute lung injury | Anecdotal report (human) [144] |
Allergic encephalomyelitis | Animal models |
Allergic rhinitis | Controlled trial (human) |
Arthritis | Anecdotal report (human) [145] |
Asthma | Animal models, controlled trials (human) |
Cancer | Animal models, some trials (human) Some meta-analyses |
Chronic obstructive pulmonary disease | Controlled trial (human) |
Delayed-type hypersensitivity reactions | Animal models |
Inflammatory bowel disease | Some controlled trials (human) |
Interstitial cystitis | Human experimental model of condition [146] Related molecule (pentosan polysulphate) used clinically [147] |
Transplant rejection | Animal m odels |
Wound healing | Various reports in animals and humans |
Heparin and Recurrent Implantation Failure (RIF)
The term RIF has been used to describe IVF treatment failure due to embryos’ failure to implant. The ESHRE PGD consortium document mentioned that RIF can be considered after more than three high-quality embryo transfers or implantation failure with transfer of ≥10 embryos in multiple transfers with exact numbers to be determined by each center [48]. A meta-analysis with systemic review of the literature compared the use of LMWH with placebo or no adjuvant treatment in women with RIF undergoing IIVF/ICSI. The results have shown that in women with at least three RIFs, the use of LMWH during IVF treatment improved the live birth rate in 79 %. These results show that heparin might have a useful and important role during IVF treatment and more research should be done.
Heparin and Recurrent Pregnancy Loss
The role of heparin and LMWH has long been investigated and published, mainly in the context of antiphospholipid syndrome and thrombophilia. As was shown in the LIVE-ENOX study, prophylactic administration of enoxaparin to women with RPL and thrombophilia was found to be effective and safe [49]. Several studies that investigated heparin’s role when it comes to women who suffer with RPL with an unknown etiology didn’t show heparin to be beneficial, despite heparin attributes described hitherto.
One research done in Israel in 2006 compared live birth rates in women who suffered from RPL. One group was administered enoxaparin (LMWH) while the other received aspirin. No statistical difference was found between the two groups when comparing live birth rates, but a difference was shown between the live birth rates as expected from the literature (60 % after three pregnancy failures, 40 % after four pregnancy failures) and their results in both groups with more than 80 % live birth rate.
In a study where enoxaparin was compared to placebo when treating women with RPL with unknown cause no significant difference in live birth rate was found between the two groups (66.6 and 72.9 %, respectively) [50].
A study that compared different treatment methods for women with unexplained RPL found no difference in live birth rate (written in brackets after each group) when comparing treatment regimens with aspirin (50.8 %), aspirin + (LMWH) nadroparin (54.5 %), and placebo (57.0 %) [51].
In conclusion, heparin has a place of honor in RPL treatment thanks to its anticoagulant ability; heparin’s additional characteristics are the reason for the conclusion that it might be useful in RPL women who don’t have thrombophilia. Hitherto, no conclusive evidence has yet supported this idea. Further research and investigation are needed to be sure that we do not inject heparin for no reason.
Involvement of Immunity in Recurrent Pregnancy Loss
Pregnancy: A Balancing Act of the Maternal Immune System
Pregnancy success requires suppression of the mother’s immune system, enabling an immune-tolerant state [52]. The maternal immune system of endometrial and decidual tissues is primarily composed of immune cell populations that are myelomonocytic, T cells and decidual Natural Killer (dNK) cells . dNK cells are thought to play an important role, serving as the predominant cell type in this process [53]. These immune cell populations play an important role in placental tissue development, fetal growth, and establishment of immune-tolerance by secretion of various type-1 cytokines (IFN-γ, TNF-α, TNF-β, and IL-2) that contribute to cellular immunity, and type-2 cytokines (IL-4, IL-5, IL-6, IL-10, and IL-13) that encourage humoral immunity.
The balance between type-1 and type-2 cytokines is essential to the success of pregnancy [54–56]. IFN-γ supports remodeling of spiral arteries, promotes immune tolerance by inhibition of pro-inflammatory TH17 cells, and encourages indoleamine 2,3-dioxygenase (IDO) upregulation in myelomonocytic cells, which in turn induces Treg FOXP3+ and suppress CD8+ T cells [53, 57, 58]. IFN-γ also increases TRAIL-R expression on syncytiotrophoblasts, and human trophoblasts express FasL; both molecules can serve as a mechanism for protection against dNK and T cells. TNF-α mediates apoptosis of cytotrophoblasts and promotes the formation of syncytiotrophoblasts [59]. IL-10 inhibits the secretion of IFN-γ and TNF-α, modulating trophoblast invasion and suppressing TH17 cells [60]. IL-10 also regulates the number of myelomonocytic cells that are the main antigen presenting cells in the decidua tissue (20–30 % throughout pregnancy) and have a role in defending against microbes. Type 3 cytokines, such as TGF-β, can regulate the balance between type-1 and type-2 cytokines. Secretion of TGF-β by decidual myelomonocytic cells leads to inhibition of dNK and prevents the killing of the cytotrophoblast by dNK [61]. Other cytokines and chemokines additionally have a role in placenta development and immune tolerance. For example, MIP-1α (CCL3) and MIP-1β (CCL4) are important to attract and activate maternal immune cells, while IL-8 promotes trophoblast migration. GM-CSF regulates decidual leukocyte populations and enhances placental growth and differentiation [62, 63] thereby, both type-1 and type-2 cytokines have a major role in regulating the development of placental tissue and establishment of an immune-tolerance towards the fetus.
Recurrent Pregnancy Loss and NK Cells: Breaking the Balance
Recurrent pregnancy loss (RPL) was recently suggested to be associated with superfertility. In a retrospective analysis of 560 RPL patients, Salker et al. showed that 40 % of the women were considered “superfertile,” relative to the prevalence in the total population that is about 3 % [21, 64]. Superfertility refers to a longer “Implantation window ” [65]. This interval in the menstrual cycle is a transient endometrial state with a high receptivity for the adherence of a blastocyst that is dependent on paracrine signals from stromal cells to the immune cell populations and is part of the decidualization process[66]. This prolonged blastocyst receptivity correlates with early pregnancy loss and superfertility [21].
During the first trimester of pregnancy, dNK cells are the dominant cell population and are about 50–70 % of leukocytes (Fig. 14.3) [67–69]. dNK cells secrete IFN-γ, TNF-α, GM-CSF, IP-10, MIP-1α (CCL3), MIP-1β (CCL4), IL-8, VEGF, PLGF, Ang-1, Ang-2, IL-10, and IL-1RA [70, 71]. Decidual stromal cells can regulate myelomonocytic cells and dNK cells by the IL-33/ST2L/sST2 axis. Upon decidualization, a mechanism that was disordered in stromal cells of women with RPL [72], the IL-33/ST2 axis, also promotes the temporal expression of receptivity genes in stromal cells; failure to restrain this axis leads to a longer implantation window [72]. IL-33 promotes proliferation of stromal cells, macrophages, and trophoblasts [73]. ST2, a receptor for IL-33, is expressed on dNK but not on peripheral blood NK cells (see Chap. 6 for elaborated information). Culture medium from decidual stromal cells inhibits the cytolytic activity of dNKs. Furthermore, it shifts the cytokines’ balance of dNK from type-1 to type-2 by downregulating the secretion of IFN-γ and TNF-α, and upregulating IL-10 [74]. In mice, administration of IFN-γ and TNF-α promote abortions and both cytokines inhibit growth of human trophoblasts in vitro [55]. IL-33 also up regulates the expression of PCNA in stromal cells, a nuclear protein that was shown to inhibit NK cell function by interaction with NKp44 [75–77].
Fig. 14.3
Human natural killer cells interacting with CFSE-labeled cervical cancer (HeLa) cells (green) and nuclei dye (DAPI blue). Filamentus actin is labeled with Phalloidin (yellow to red) to show points of interaction. Acquires using FV1000 (Olympus) equipped with 100× oil objective. [Confocal Microscopy, Courtesy of Mr. Uzi Hadad, Ben-Gurion University of the Negev]
Moreover, it was shown that decidual stromal cells and trophoblasts express ligands to DNAM-1, NKp30, and NKp44, which are activation receptors expressed on NK cells [71, 78]. Recently, it was suggested that women with RPL have impaired regulation of pregnancy-related cytokines [79, 80]. Analysis of peripheral blood NK cells and their NKp30, NKp44, and NKp46 receptors (NCRs) expression compared to intracellular cytokine expression (TNF-a, IFN-γ, IL-4, IL-10) after activation revealed a negative correlation between NCRs’ protein expression and intracellular cytokine. Furthermore, the correlation between the mRNA expression of NKp30, NKp46, and pregnancy-related cytokines seems to be lost in placental tissue from RPL patients compared to elective abortions. Moreover, mRNA of IFN-γ, TNF-α, and IL-10 was higher in placenta tissue obtained from spontaneous miscarriage patients [81, 82].
Depletion of NK cells in a murine model did not have an effect on the outcome of pregnancy [83]. However, in an IL-10 KO murine model, NK cell activity was enhanced after LPS administration and led to pregnancy loss. Depletion of dNK or administration of anti-TNF-α or -IL-10 rescued pregnancies [84]. These observations point to the necessity of a balance between type-1 and type-2 cytokines, which in turn influence dNK cells activity and can lead to pregnancy loss [85].
Influencing the Balance: Target for Therapy
Immunologic etiologies can be attributed to 40 % of all RPL cases. There is a strong association between pregnancy loss and type-1 cytokines while a successful pregnancy is associated with type-2 cytokines [54–56]. In a murine model of antiphospholipid syndrome (APS) , antiphospholipid (aPL) Abs increased in decidual and systemic TNF-α levels, which promote trophoblast apoptosis, identifying TNF blockade as a potential therapy for the pregnancy complications [86]. TNF-α activity can be blocked with monoclonal antibodies against the TNF-α molecule (adalimumab) or against soluble TNF-α receptors (etanercept) [87, 88]. TNF-α blockers administered with or without anticoagulants, or anticoagulants + IVIG treatments (control groups), were given to women with RPL history during pregnancy. The live birth rate of patients who received TNF-α blockers was 71 %, whereas the control groups showed rates of 19 and 54 %, respectively [89]. The same trend was observed in women undergoing IVF; TNF-α blockers improve the implantation rate [90].
G-CSF was also found to have a positive effect on RPL patients. G-CSF reduces the cytotoxicity and IFN-γ secretion of dNK cells, increases the number of Treg cells, and reduces the synthesis of various cytokines, among them TNF-α [91]. A randomized controlled trial of women with RM treated with G-CSF or a placebo showed that G-CSF administrations increased the live birth rate from 48.5 % (placebo group) to 82.8 % (G-CSF group) [92]. In a second study, treatment of RPL patients undergoing assisted reproductive treatment (IVIG, LMWH, cortisone) with G-CSF increased the live birth rate from 13 to 32 % [93].
The results of these few clinical studies reveal the necessity of maintaining a balance between type 1 and type 2 cytokines in early pregnancy. TNF-α inhibition seems to have much potential in future clinical therapies.
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