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Role of chemokines in the endometrium and in embryo implantation
Abstract
Chemokines are well known for their roles in the immune system; convincing evidence has emerged demonstrating a broader role for chemokines in the endometrium, particularly during embryo implantation. This review highlights the evidence on newly defined roles for chemokines in the endometrium during embryo implantation, with particular focus on those chemokines expressed by the endometrium.
The highly regulated temporal and spatial expression of chemokines in the endometrium leads not only to specific recruitment and activation of appropriate leucocytes but also coordinates the precisely orchestrated invasion of trophoblasts through the decidua and maternal vasculature. Results to date implicate chemokine signalling at the maternal-foetal interface in important processes during implantation and placentation, such as leucocyte recruitment and controlled trophoblast invasion. Unravelling such actions of chemokines in the endometrium has provided new insights into these complex processes.
Disturbances of chemokine production, processing, or actions are likely to contribute to dysfunction of implantation and placentation, with implications for early pregnancy loss and disturbed placental and foetal development. More research into altered chemokine function in such conditions may provide leads for new clinical interventions.
... Chemokines are a superfamily of small-molecule cytokines, widely expressed in trophoblast cells, decidual stromal cells (DSCs), and decidual immune cells (DICs) at the maternal-fetal interface. Emerging evidence has identified chemokines and chemokine receptors as essential contributors in pregnancy, participating in trophoblast invasion, decidualization, and immune cell recruitment (Hannan and Salamonsen, 2007;Ramhorst et al., 2016;Ashley et al., 2021). Moreover, the aforementioned functional abnormalities of chemokines have been reported in several pregnancy complications, including preeclampsia (PE), recurrent spontaneous abortion (RSA), and preterm birth (PTB) (Whitcomb et al., 2007;Kwak et al., 2014;Ali et al., 2021;Wang et al., 2021;Zheng et al., 2021). ...
... At the maternal-fetal interface, the trophoblast cells, DSCs, and DICs establish rich chemokines and chemokine receptor network (Du et al., 2014). This chemokine network not only regulates specific recruitment and activation of appropriate leucocytes but also coordinates precisely orchestrated invasion of trophoblast through the decidua and maternal vasculature (Jones et al., 2004;Red-Horse et al., 2004;Hannan and Salamonsen, 2007;Fraccaroli et al., 2009). In this section, we will provide a detailed overview of the regulation and function of crucial chemokines at the maternal-fetal interface, as shown in Figure 1. ...
... The abnormal expression of chemokines and chemokine receptors can interrupt the trophoblast function, uterus angiogenesis, and maternal-fetal immune tolerance, thereby participating in pregnancy complications (Hannan and Salamonsen, 2007). In this part, we will focus on the relationship between chemokines and pregnancy-associated diseases, including PE, RSA, and PTB. ...
Successful pregnancy requires the maternal immune system to tolerate the semi-allogeneic embryo. A good trophoblast function is also essential for successful embryo implantation and subsequent placental development. Chemokines are initially described in recruiting leukocytes. There are rich chemokines and chemokine receptor system at the maternal–fetal interface. Numerous studies have reported that they not only regulate trophoblast biological behaviors but also participate in the decidual immune response. At the same time, the chemokine system builds an important communication network between fetally derived trophoblast cells and maternally derived decidual cells. However, abnormal functions of chemokines or chemokine receptors are involved in a series of pregnancy complications. As growing evidence points to the roles of chemokines in pregnancy, there is a great need to summarize the available data on this topic. This review aimed to describe the recent research progress on the regulation and function of the main chemokines in pregnancy at the maternal–fetal interface. In addition, we also discussed the potential relationship between chemokines and pregnancy complications.
... The presence and mixture of immune cells at the implantation site is regulated by chemokines, being increasingly recognized as important regulators of endometrial function due to their chemotactic activity [16][17][18]. CCL (CC-chemokine ligand) 5 and CCL2 belong to the C-C subfamily of chemokines, which mainly attract and activate monocytes/macrophages, T and NK cells [19]. In the human endometrium, CCL5 and CCL2 show specific cyclic patterns of expression and have a strong influence on the immune milieu at the feto-maternal interface [16,17,20,21]. ...
... CCL (CC-chemokine ligand) 5 and CCL2 belong to the C-C subfamily of chemokines, which mainly attract and activate monocytes/macrophages, T and NK cells [19]. In the human endometrium, CCL5 and CCL2 show specific cyclic patterns of expression and have a strong influence on the immune milieu at the feto-maternal interface [16,17,20,21]. ...
... Bars represent mean ± SEM; *p < 0.05 compared to untreated cells, # p < 0.05 compared to "stimulation" endometrial receptivity and implantation success [4,34]. As chemokines are essential regulators of the immune cell milieu in the human endometrium [17,18], the strong induction of CCL5 and CCL2 by the combination of TNF-α plus IFN-γ might be a stimulatory feedback-signal for the recruitment of leukocytes under pro-inflammatory conditions related to implantation disorders. Consequently, TNF-α and IFN-γ are not only sensitizers for Fas-mediated apoptosis in the human endometrium [14], but also indirectly mediate chemo-attraction of immune cells, which in turn further stimulate this pro-inflammatory and pro-apoptotic loop leading to cellular breakdown und disturbance of the subtle balanced local immune milieu. ...
Purpose:
Tumor necrosis factor (TNF)-α and interferon (IFN)-γ are pro-inflammatory cytokines which have been shown to be involved in the pathophysiology of implantation disorders. Both cytokines in combination are able to sensitize primarily resistant human endometrial stromal cells (ESCs) to Fas-induced apoptosis. Since CCL (CC-chemokine ligand) 5 and CCL2 are important regulators of the endometrial immune cell population, we examined the impact of TNF-α and IFN-γ on these two chemokines under non-apoptotic and apoptotic conditions.
Methods:
ESCs were isolated from hysterectomy specimens, decidualized in vitro and incubated with TNF-α, IFN-γ, an activating anti-Fas antibody and a caspase-inhibitor. CCL5 and CCL2 were measured using ELISA and real-time RT-PCR. Apoptosis was determined by flow cytometry, and cellular viability and membrane integrity were measured by fluorescent assays.
Results:
The secretion of CCL5 and CCL2 was stimulated in undifferentiated and decidualized ESCs by the combination of TNF-α and IFN-γ under non-apoptotic as well as apoptotic (with Fas-stimulation in parallel) conditions. TNF-α or IFN-γ alone did not have this effect. The stimulatory influence of TNF-α plus IFN-γ on CCL5 and CCL2 in ESCs was also seen on the transcriptional level. Inhibition of cell death by a caspase-inhibitor had no influence on the secretion of CCL5 and CCL2 in ESCs under apoptotic stimulation.
Conclusion:
TNF-α and IFN-γ modulate the secretion of chemokines in ESCs independently of Fas-induced apoptosis. These results suggest a constant response pattern on pro-inflammatory cytokines within the population of human ESCs.
... Further research in this area revealed that use of ISGs as a marker of early pregnancy diagnosis had variable effectiveness (Han et al. 2006, Stevenson et al. 2007, for example, OAS1 mRNA levels were successfully used to assess pregnancy on 18 d in heifers but not in cows (Green et al. 2010). Few investigations (Hannan andSalamonsen 2007, Sakumoto et al. 2017) have indicated role of the chemokines as important players in the process of embryonic implantation. Since, successful embryonic implantation is a well coordinated process involving maternal-embryonic communication by chemokine signaling and extensive endometrial modeling (Hannan andSalamonsen 2007, Kiefer andSiekmann 2011), functional involvement of chemokines cannot be denied. ...
... Few investigations (Hannan andSalamonsen 2007, Sakumoto et al. 2017) have indicated role of the chemokines as important players in the process of embryonic implantation. Since, successful embryonic implantation is a well coordinated process involving maternal-embryonic communication by chemokine signaling and extensive endometrial modeling (Hannan andSalamonsen 2007, Kiefer andSiekmann 2011), functional involvement of chemokines cannot be denied. One study has reported increased expression of these chemokines in peripheral blood leucocytes (PBLs) of cows during peri-implantation period (Sakumoto et al. 2018). ...
The present investigation was aimed to evaluate novel implication of chemokine genes CXCL10 (C-X-C motif chemokine 10) and CCL 8 (C-C motif chemokine 8) genes for use as ideal pregnancy biomarker in dairy buffaloes. We studied expression profile of both these chemokine genes in whole blood of Murrah buffaloes on day 12, 15, 18 and 21 post artificial insemination (AI) using SYBR green chemistry based quantitative real time reverse transcription PCR. Our investigations revealed a consistent increase in transcriptional abundance of CCL8 and CXCL10 genes during this period, although the difference in expression level was not significant between day 15 and 18 post AI for CXCL10 gene. We also studied the effect of parity on the expression profile of these two genes and found that expression level of both these genes is independent of parity status of the animal. Based on the results, it can be concluded that these chemokine genes can be used as early pregnancy biomarker on any day between days 12 to 21 post artificial insemination in buffaloes irrespective of their parity status.
... Chemokines are a family of chemotactic cytokines that recruit and activate leukocytes from the periphery to the site of inflammation [23]. In the uterus, chemokines act as chemo-attractants for different leukocytes-uterine natural killer cells (uNK), T-cells, and macrophages [24]. ...
... This can cause abnormal development of spiral arteries and inadequate blood flow to the feto-placental unit, which leads to oxidative stress to the developing trophoblast and implantation failure or miscarriage [28]. Besides chemotactic properties, chemokines also alter the expression of cellular adhesion molecules and extracellular matrix (ECM) components that allow cell migration and it has been shown that they have functional effects on trophoblast cell attachment and migration [24]. Genes SDF1(CXCL12) and its receptor CXCR4, both part of chemokine signaling pathway and up-regulated in non-PG endometrium in the present study, are known to play a role in the cross-talk between trophoblast and endometrium, recruitment of lymphocytes into the uterus of pregnant females, and vascularization [29]. ...
Obesity and being overweight are growing worldwide health problems that also affect women of reproductive age. They impair women’s fertility and are associated with lower IVF success rates. The mechanism by which increased body weight disrupts fertility has not yet been established. One possibility is that it affects the process of embryo implantation on the endometrial level. The purpose of our study was to determine the differences in enriched biological pathways in the endometrium of overweight and obese women undergoing IVF procedures. For this purpose, 14 patients (5 pregnant, 9 non-pregnant) were included in the study. Endometrial samples were obtained during the window of implantation and RNA sequencing was performed. There were no differences in general patient’s and IVF cycle characteristics between pregnant and non-pregnant women. In the endometrial samples of women who did not conceive, pathways related to the immune response, inflammation, and reactive oxygen species production were over-expressed. Our findings show that the reason for implantation failure in overweight and obese women could lie in the excessive immune and inflammatory response at the endometrial level.
... 21 The abundance, phenotype and function of these immune cell networks are modulated by a complex array of pro-inflammatory and immunemodulatory cytokines and chemokines in the uterine endometrium. [22][23][24] Shifts in the endometrial cytokine balance can impact the maternal immune response and modify the events of placental development, to either promote or compromise progression of a viable pregnancy. 3 Treg cell-mediated tolerance arises in the preimplantation phase of early pregnancy and depends on interactions between maternal, paternal and conceptus-derived signals at the mucosal surface of the uterine endometrium. ...
... 3,22,23 The cytokines that were elevated after Intralipid infusion each has pro-inflammatory and immuneregulatory properties, and all are expressed in the endometrium during early pregnancy. 22,24,57 This local inflammatory state is accompanied by systemic indicators of inflammatory activation, as evidenced by higher plasma CRP levels in pregnant than in non-pregnant IVF patients detectable from 10 days after embryo transfer. 58 Elevated CRP associated with implantation success was not detected in the current study, presumably because of the earlier sampling time, and the small number of women evaluated. ...
Objectives:
Intravenous infusion of Intralipid is an adjunct therapy in assisted reproduction treatment (ART) when immune-associated infertility is suspected. Here, we evaluated the effect of Intralipid infusion on regulatory T cells (Treg cells), effector T cells and plasma cytokines in peripheral blood of women undertaking IVF.
Methods:
This prospective, observational pilot study assessed Intralipid infusion in 14 women exhibiting recurrent implantation failure, a clinical sign of immune-associated infertility. Peripheral blood was collected immediately prior to and 7 days after intravenous administration of Intralipid. Plasma cytokines were measured by Luminex, and T-cell subsets were analysed by flow cytometry.
Results:
A small increase in conventional CD8+ T cells occurred after Intralipid infusion, but no change was seen in CD4+ Treg cells, or naïve, memory or effector memory T cells. Proliferation marker Ki67, transcription factors Tbet and RORγt, and markers of suppressive capacity CTLA4 and HLA-DR were unchanged. Dimensionality-reduction analysis using the tSNE algorithm confirmed no phenotype shift within Treg cells or other T cells. Intralipid infusion increased plasma CCL2, CCL3, CXCL8, GM-CSF, G-CSF, IL-6, IL-21, TNF and VEGF.
Conclusion:
Intralipid infusion elicited elevated pro-inflammatory cytokines, and a minor increase in CD8+ T cells, but no change in pro-tolerogenic Treg cells. Notwithstanding the limitation of no placebo control, the results do not support Intralipid as a candidate intervention to attenuate the Treg cell response in women undergoing ART. Future placebo-controlled studies are needed to confirm the potential efficacy and clinical significance of Intralipid in attenuating cytokine induction and circulating CD8+ T cells.
... During implantation, there is a highly regulated temporal and spatial expression of chemokines in the endometrium. This leads not only to specific recruitment and activation of neutrophils but also coordinates proper placentation and angiogenesis (Hannan and Salamonsen, 2007). Up-regulation in the activity of CD62L, CD11b and IL-8 in NP cows at time of initiation of implantation indicates hostile condition for implanting embryo and thus, may influence the outcome of embryo implantation. ...
... TNF␣ also induces PGF2␣ production and hence uterine contraction which counteracts pregnancy (Singh et al., 2008). According to Hannan and Salamonsen (2007), any disturbances of chemokine production, processing, or actions are likely to contribute to dysfunction of implantation and placentation, with implications for early pregnancy loss and disturbed placental and foetal development. Pro-inflammatory cytokines induce the rapid expression of selectins on endothelial cell plasma membrane which is responsible for neutrophil adhesion and transmigration (Dominguez et al., 2005). ...
... The primary function of chemokines is to command immune cell migration into infected or inflamed tissue to initiate an effective immune response [42]. They also play a role in angiogenesis and hematopoiesis, and regulate activation, proliferation, differentiation, and apoptosis in the cells they attract [43,44]. Functional chemokines and their receptors are widely expressed in maternal-conceptus tissues and are a major player in tissue communication and pregnancy success [45]. ...
... The primary function of chemokines is to command immune cell migration into infected or inflamed tissue to initiate an effective immune response [42]. They also play a role in angiogenesis and hematopoiesis, and regulate activation, proliferation, differentiation, and apoptosis in the cells they attract [43,44]. Functional chemokines and their receptors are widely expressed in maternal-conceptus tissues and are a major player in tissue communication and pregnancy success [45]. ...
It is possible to identify sub-populations of sows in every pig herd that consistently give birth to low birth weight (BW) piglets, irrespective of the litter size. A previous study from our group demonstrated that placental development is a main factor affecting the litter birth weight phenotype (LBWP) in sows, thereby impacting the BW of entire litters, but the biological and molecular pathways behind this phenomenon are largely unknown. The aim of this study was to investigate the differential gene expression in placental tissues at day 30 of gestation between low LBWP (LLBWP) vs. high LBWP (HLBWP) sows from a purebred Large White maternal line. Using mRNA sequencing, we found 45 differentially expressed genes (DEGs) in placental tissues of LLBWP and HLBWP sows. Furthermore, (GO) enrichment of upregulated DEGs predicted that there were two biological processes significantly related to cornification and regulation of cell population proliferation. To better understand the molecular interaction between cell proliferation and cornification, we conducted transcriptional factor binding site (TFBS) prediction analysis. The results indicated that a highly significant TFBS was located at the 5′ upstream of all four upregulated genes (CDSN, DSG3, KLK14, KRT17), recognized by transcription factors EGR4 and FOSL1. Our findings provide novel insight into how transcriptional regulation of two different biological processes interact in placental tissues of LLBWP sows.
... Relative expression for each mRNA transcript was normalized to the expression of the GAPDH transcript and are reported as means ± SEM relative to CT (= 1.0). Different superscript letters indicate differences in each treatment of CCL2 for each panel (P < 0.05) decidua during pregnancy (Hannan and Salamonsen 2007). However, there was a lack of information on the regulation mechanism of the expression of CCL2 in bovine endometrial cell types during pregnancy. ...
C-C motif chemokine ligand 2 (CCL2) has been reported to be expressed in the bovine endometrium during pregnancy. However, the details of its functions involved in the implantation mechanism are still not clear. The purpose of this study is to analyze the functional properties of CCL2 in the bovine endometrium and embryos. The expression of CCR2 was not different between the luteal phase and implantation phase of their endometrial tissues, but was significantly high in IFNa treated bovine endometrial stromal (BES) cells in vitro. The expressions of PGES1, PGES2, AKR1C4, and AKR1C4 were high at the implantation stage compared with the luteal stage. On the other hand, PGES2 and AKR1B1 in BEE and PGES3 and AKR1A1 in BES were significantly increased by CCL2 treatment, respectively. The expressions of PCNA and IFNt were found significantly high in the bovine trophoblastic cells (BT) treated with CCL2 compared to the control. CCL2 significantly increased the attachment rate of BT vesicles to BEE in in vitro co-culture system. The expression of OPN and ICAM-1 increased in BEE, and ICAM-1 increased in BT by CCL2 treatment, respectively. The present results indicate that CCL2 has the potential to regulate the synthesis of PGs in the endometrium and the embryo growth. In addition, CCL2 has the possibility to regulate the process of bovine embryo attachment to the endometrium by modulation of binding molecules expression.
... The endometrium expresses chemokines in a highly regulated temporal and spatial pattern during implantation. This not only leads to precise neutrophil recruitment and activation but also appropriate placentation and angiogenesis coordination [38]. Upregulation of CD62L and CD11b expression in NP cows at the time of implantation was observed. ...
abstract
Pregnancy is a complicated physiological process that involves synchronized coordination between
immune and endocrine systems. Neutrophils have been suggested as a critical immune cell for embryo
implantation and pregnancy maintenance. The present study was conducted to evaluate the dynamic
changes in the mRNA expressions of the cluster of designation (CD11b, CD31, CD44 and CD62L) mole�cules and interferon-stimulated genes (ISG15, MX1 and OAS1) in blood neutrophils throughout preg�nancy in dairy cows and correlate them with the outcome of pregnancy. Blood samples were taken from
negative control (NC) group, and non-pregnant (NP) group at the time of artificial insemination (AI, day
zero) and on days 10, 14, 16, 18, and 21 post-AI. In pregnant (P) cows, samples were taken as described
above and after every 30 days until the time of parturition. In aborted cows, samples were collected until
the time of the abortion. Comparison between pregnant, non-pregnant and aborted cows revealed that
the expression of CD molecules increased (p < 0.05) on days 14, 16, 18 and 21 post-AI only in NP cows as
compared to other groups. Although the expression of CD molecules remained constant throughout the
study period in pregnant and aborted cows, the expression of CD11b, CD31 and CD62L increased
(p < 0.05) on the day of abortion and parturition. Unlike CD molecules, the expression of CD44 decreased
significantly (p < 0.05) at the time of abortion. There was a significant (p < 0.05) increase in the
expression of interferon-stimulated genes including MX1, OAS1 and ISG15 during the peri-implantation
period in pregnant cows, and at the time of abortion in aborted cows. However, the expression of ISGs
was lower (p < 0.05) in non-pregnant cows as compared to the other groups. The results revealed the
critical role played by neutrophils during pregnancy and form the basis to unravel the underlying
mechanism for neutrophil associated immunological infertility in bovines.
... IL-8 (CXCL8) is released by NK cells implying the migration of trophoblast cells for endovascular invasion and maternal vascular remodeling [102]. Elevated levels of cord blood IL-8 have been associated with pre-eclampsia [103] and moderate-severe bronchopulmonary dysplasia in NBs [48]. ...
Chronic venous disease (CVD) is a common vascular disorder characterized by increased venous hypertension and insufficient venous return from the lower limbs. Pregnancy is a high-risk situation for developing CVD. Approximately a third of the women will develop this condition during pregnancy, and similarly to arterial hypertensive disorders, previous evidence has described a plethora of alterations in placental structure and function in women with pregnancy-induced CVD. It is widely known that arterial-induced placenta dysfunction is accompanied by an important immune system alteration along with increased inflammatory markers, which may provide detrimental consequences for the women and their offspring. However, to our knowledge, there are still no data collected regarding cytokine profiling in women with pregnancy-induced CVD. Thus, the aim of the present work was to examine cytokine signatures in the serum of pregnant women (PW) with CVD and their newborns (NB). This study was conducted through a multiplex technique in 62 PW with pregnancy-induced CVD in comparison to 52 PW without CVD (HC) as well as their NB. Our results show significant alterations in a broad spectrum of inflammatory cytokines (IL-6, IL-12, TNF-α, IL-10, IL-13, IL-2, IL-7, IFN-γ, IL-4, IL-5, IL-21, IL-23, GM-CSF, chemokines (fractalkine), MIP-3α, and MIP-1β). Overall, we demonstrate that pregnancy-induced CVD is associated with a proinflammatory environment, therefore highlighting the potentially alarming consequences of this condition for maternal and fetal wellbeing.
... Several chemokines are produced throughout the estrous cycle and pregnancy, playing essential roles in regulating endometrial functions for estrous cyclicity and establishing and maintaining pregnancy [48][49][50]. The atypical chemokine receptors (ACKRs) are similar to traditional chemokine receptors but regulate chemokine activity by limiting their spatial availability or removing them from in vivo sites [51]. ...
Uterine homeostasis is maintained after mating by eliminating pathogens, foreign cells, and proteins by a transient inflammation of the uterus. Such inflammation does not occur in the oviductal sperm reservoir (utero-tubal junction, UTJ), colonized by a population of potentially fertile spermatozoa before the inflammatory changes are triggered. Semen entry (spermatozoa and/or seminal plasma) modifies the expression of regulatory genes, including cell proliferation and differentiation-related transcripts. Considering pigs display a fractionated ejaculation, this study aims to determine whether different ejaculate fractions differentially modulate cell proliferation and differentiation-related transcripts in the sow reproductive tract during the peri-ovulatory stage. Using species-specific microarray analyses, the differential expression of 144 cell proliferation and differentiation-related transcripts was studied in specific segments: cervix (Cvx), distal and proximal uterus (DistUt, ProxUt), UTJ, isthmus (Isth), ampulla (Amp), and infundibulum (Inf) of the peri-ovulatory sow reproductive tract in response to semen and/or seminal plasma cervical deposition. Most mRNA expression changes were induced by mating. In addition, while mating upregulates the fibroblast growth factor 1 (FGF1, p-value DistUt = 0.0007; ProxUt = 0.0253) transcript in the endometrium, both its receptor, the fibroblast growth factor receptor 1 (FGFR1, p-value DistUt = 2.14 e−06; ProxUt = 0.0027; UTJ = 0.0458) transcript, and a potentiator of its biological effect, the fibroblast growth factor binding protein 1 (FGFBP1), were downregulated in the endometrium (p-value DistUt = 0.0068; ProxUt = 0.0011) and the UTJ (p-value UTJ = 0.0191). The FGFBP1 was downregulated in the whole oviduct after seminal depositions (p-value Isth = 0.0007; Amp = 0.0007; Inf = 6.87 e−05) and, interestingly, FGFR1 was downregulated in the endometrium in the absence of semen (p-value DistUt = 0.0097; ProxUt = 0.0456). In conclusion, the findings suggest that spermatozoa, seminal components, and the act of mating trigger, besides inflammation, differential mechanisms in the peri-ovulatory female reproductive tract, relevant for tissue repair.
... This process is triggered by the postovulatory rise in progesterone that increases intracellular cAMP levels. This causes EnSCs to transition from spindleshaped fibroblasts to epithelioid-like decidual cells that secrete increased levels of insulin-like growth factor binding protein 1 (IGFBP-1) and prolactin, 2 classic decidualization markers (14), as well as a range of chemokines and cytokines that coordinate changes in the local immune cell composition, contribute to structural changes in the uterine epithelium, and regulate trophoblast invasion (14,15). Thus, aberrant decidualization can predispose a woman to pregnancy failure or associated obstetric complications. ...
Objective
Miscarriage affects 1 in 7 pregnancies, and antiphospholipid autoantibodies (aPLs) are one of the biggest risk factors for recurrent pregnancy loss. While aPLs target the endometrial stroma, little is known about their impact. Endometrial stromal cells (EnSCs) undergo decidualization each menstrual cycle, priming the uterus to receive implanting embryos. Thus, appropriate decidualization and EnSC function is key for establishment of a successful pregnancy. This study was undertaken to explore the effects of aPL on EnSC decidualization, senescence, and inflammation.
Methods
EnSCs under decidualizing conditions were exposed to aPL or control IgG alone or in the presence of either a Toll‐like receptor 4 (TLR‐4) antagonist, a p38 MAPK inhibitor, a reactive oxygen species (ROS) inhibitor, low molecular weight heparin (LMWH), or acetyl salicylic acid. Secretion of decidualization markers and inflammatory interleukin‐8 were quantified by enzyme‐linked immunosorbent assay, and senescence‐associated β‐galactosidase activity was evaluated. In a mouse model of decidualization, aPL or control IgG was administered, and uterine expression levels of decidualization and inflammatory markers were quantified by real‐time quantitative polymerase chain reaction.
Results
Antiphospholipid antibodies increased human EnSC decidualization, senescence, and inflammation. This phenotype was recapitulated in the mouse model. The decidualization and inflammatory responses were partially mediated by TLR‐4 and p38 MAPK, while the decidualization and senescence responses were ROS‐dependent. LMWH, commonly used to treat aPL‐positive women at risk of obstetric complications, reduced the ability of aPL to increase EnSC decidualization and inflammation.
Conclusion
These findings shed new light on the pathogenesis of pregnancy complications in women with aPLs and underscore the benefit of heparin in preventing pregnancy loss in this high‐risk population.
... During these phases, PMNs represent the most common phagocytic cells in the uterus (Skjerven, 1956). In pregnancy, PMNs can detect implantation (Kizaki et al., 2013;Manjari et al., 2016;Shirasuna et al., 2012), causing neutrophils to be recruited and activated in a specific way allowing proper placentation and angiogenesis to occur (Hannan and Salamonsen, 2007). Furthermore, PMNs showed a slight decrease in number and activity in successful pregnancy implantation. ...
The interaction between early embryo and maternal immune system for the establishment of pregnancy is the focus of several studies; however, it remains unclear. The maternal immune response needs to keep a balance between avoiding any damage to the conceptus and maintaining its function in combating microbes as well. When conceptus-maternal crosstalk cannot achieve this balance, pregnancy losses might occur. Intercommunication between mother and conceptus is fundamental during early pregnancy to dictate the outcome of pregnancy. In ruminants, the embryo reacts with the maternal system mainly via interferon tau (IFNT) release. IFNT can act locally on the embryo and endometrial cells and systemically in several tissues and cells to regulate their response via the expression of interferon-stimulated genes (ISGs). Also, IFNT can induce the expression of inflammatory-related genes in immune cells. Day 7 embryo induces a shift in the maternal immune response towards anti-inflammatory (Th2) immune responses. During maternal recognition of pregnancy, peripheral mononuclear cells (PBMCs) and polymorphonuclear cells (PMNs) express markers that configure an anti-inflammatory response. However, PMNs response is more sensitive to the effects of IFNT. PMNs are more likely to express interferon-stimulated genes (ISGs), transforming growth factor-beta (TGFB), interleukin 10 (IL10), and arginase-1 (ARG1), configuring one of the most rapid immune responses to early pregnancy. This review focus on the local and peripheral immune responses during early pregnancy in ruminants, mainly the PMNs function in the immune system.
... Via modification of leukocyte trafficking, chemokines play a critical role in the establishment of pregnancy and its maintenance. Attracted by chemokines, leukocyte subsets affect placental angiogenesis, immune tolerance and invasion of trophoblasts [12,13]. During parturition, chemokines participate in the secretion of MMPs and promote uterine contractions and the release of prostaglandin E (PGE) [4,11,14]. ...
Physiological parturition is characterized by sterile, inflammatory-like processes. During parturition, the placenta expresses various proinflammatory mediators, such as chemokines and IL-17. Nevertheless, inflammatory processes present in the parturient mare are poorly characterized. The aim of this study was to investigate the expression of selected chemokines and IL-17 in the allantochorion and the endometrium of mares that retained fetal membranes (RFM) and expelled them physiologically. We hypothesized that the expression of these mediators may be altered in the placenta of mares with RFM and result in RFM occurrence.
Differences in mRNA expression in the placenta of investigated groups of mares were detected for CCL2, CCL3, CCL4, CCL8, CXCL1, CXCL8, CXCL10, CX3CL1 and IL-17. There were no differences in mRNA expression of CCL5 and CXCL6. Gene ontology network analysis showed enrichment in genes related to leukocyte migration, cell chemotaxis and response to chemokine in tissues of RFM mares. Analysis of association network suggested denotations between CXCL6, CXCL8, CXCL1, CCL5, CCL4, CX3CL1 and CXCL10. Moreover, possible inhibition of CXCL10 by IL-17A and prostaglandin peroxide synthase 2 (PTGS2) by CXCL1 was detected.
Our results suggest that, based on differences in chemokines and IL-17 expression, recruited subsets of leukocytes might differ between the analyzed groups of mares, which in turn may impair the separation of fetal membranes in the group of RFM mares. In addition, the results of the expression analysis suggest that macrophages might be one of the most abundant cells infiltrating the equine placenta during the expulsion of fetal membranes. Furthermore, we suspect that the synthesis of PTGS2 might be inhibited in mares with RFM.
... Cytokines are a family of secreted immune modulators controlling the function and differentiation of both immune and non-immune cells [17,18]. During pregnancy, cytokines in the internal female genital tract are crucial for the establishment of a network of communication at the maternal-fetal interface; ultimately affecting the success of implantation, placentation, and the maternal immune tolerance to the allograft constituted by the embryos/fetuses and their extraembryonic membranes [19,20]. The immunological success of allograft tolerance depends upon the establishment of a balance between pro-and anti-inflammatory cytokines [21]. ...
Pig embryo transfer (ET) is burdened by high embryo mortality, with cytokines playing a significant role in recruitment of immune cells during embryo attachment and placentation. We hereby tested if their levels in endometrium and placenta from sows carrying hemi-allogeneic (artificially inseminated sows; C+ positive control) or allogeneic embryos (sows subjected to ET; ET) during peri-implantation (D18) or post-implantation (D24) are suitable mirrors of embryo rejection or tolerance after ET. Non-pregnant sows (C-) were used as negative controls. A set of cytokines was assayed in the tissues through multiplexed microsphere-based flow cytometry (Luminex xMAP, Millipore. USA). Fewer (58.7%. p < 0.003) conceptuses were recovered at D24 after ET compared to C+ (80.9%); with more than 20% of the ET conceptuses being developmentally delayed. Cytokine levels shifted during implantation. Anti-inflammatory IL-10 levels were significantly (p < 0.05) lower in ET sows compared to C+ at D24 of pregnancy. The C+ controls (carrying hemi-allogeneic embryos) consistently showed higher levels of pro-inflammatory TNF-α, IFN-γ, and IL-2 cytokines at D18 and IL-1α at D24, compared to the ET group. This clear dysregulation of pro- and anti-inflammatory cytokine levels in sows subjected to ET could be associated with an impaired maternal immune tolerance, explaining the high embryonic mortality of ET programs.
... On the maternal side of the maternal-fetal interface, the local stromal environment, known as the decidua, is an immunologically active site where decidual stromal cells co-exist with maternal immune cells in a tightly regulated relationship that evolves as pregnancy progresses. The importance of the decidua in regulating the chronology of immune adaptations during pregnancy has been extensively reviewed elsewhere [9,66,81,91,98,118] and is summarized in Fig. 3. Here, we will focus on maternally derived endocrine, metabolic and microbial immunological pacemakers-elements that can be readily assessed during pregnancy in the peripheral blood. ...
Preterm birth is the leading cause of mortality in children under the age of five worldwide. Despite major efforts, we still lack the ability to accurately predict and effectively prevent preterm birth. While multiple factors contribute to preterm labor, dysregulations of immunological adaptations required for the maintenance of a healthy pregnancy is at its pathophysiological core. Consequently, a precise understanding of these chronologically paced immune adaptations and of the biological pacemakers that synchronize the pregnancy “immune clock” is a critical first step towards identifying deviations that are hallmarks of peterm birth. Here, we will review key elements of the fetal, placental, and maternal pacemakers that program the immune clock of pregnancy. We will then emphasize multiomic studies that enable a more integrated view of pregnancy-related immune adaptations. Such multiomic assessments can strengthen the biological plausibility of immunological findings and increase the power of biological signatures predictive of preterm birth
... As mentioned before, the whole-tissue RNA-seq analysis did not pick up significant differences for any of these factors; specifically and respectively, adjusted P-values were 0.97, 0.83 and 0.97. In general, chemokines are related to remodelling, reepithelialization and proliferation of the endometrium (Hannan and Salamonsen, 2007) and are required to maintain the feto-maternal interface as immune-privileged (Fraccaroli et al., 2009). In particular, MCP-1, SDF-1α and eotaxin are key molecules that modulate migration and infiltration of monocytes/macrophages, EnSCs and perivascular cells, respectively (Kelly et al., 2001;Park and Yang, 2011). ...
Study question:
Does an early proliferative phase endometrial biopsy harvested during ovarian stimulation harbour information predictive of the outcome following fresh embryo transfer (ET) in that same cycle?
Summary answer:
Transcriptome analysis of the whole-tissue endometrium did not reveal significant differential gene expression (DGE) in relation to the outcome; however, the secretome profile of isolated, cultured and in vitro decidualized endometrial stromal cells (EnSCs) varied significantly between patients who had a live birth compared to those with an implantation failure following fresh ET in the same cycle as the biopsy.
What is known already:
In the majority of endometrial receptivity research protocols, biopsies are harvested during the window of implantation (WOI). This, however, precludes ET in that same cycle, which is preferable as the endometrium has been shown to adapt over time. Endometrial biopsies taken during ovarian stimulation have been reported not to harm the chances of implantation, and in such biopsies DGE has been observed between women who achieve pregnancy versus those who do not. The impact of the endometrial proliferative phase on human embryo implantation remains unclear, but deserves further attention, especially since in luteal phase endometrial biopsies, a transcriptional signature predictive for repeated implantation failure has been associated with reduced cell proliferation, possibly indicating proliferative phase involvement. Isolation, culture and in vitro decidualization (IVD) of EnSCs is a frequently applied basic research technique to assess endometrial functioning, and a disordered EnSC secretome has previously been linked with failed implantation.
Study design, size, duration:
This study was nested in a randomized controlled trial (RCT) investigating the effect of endometrial scratching during the early follicular phase of ovarian stimulation on clinical pregnancy rates after IVF/ICSI. Of the 96 endometrial biopsies available, after eliminating those without fresh ET and after extensive matching in order to minimize the risk of potential confounding, 18 samples were retained to study two clinical groups: nine biopsies of patients with a live birth versus nine biopsies of patients with an implantation failure, both following fresh ET performed in the same cycle as the biopsy. We studied the proliferative endometrium by analysing its transcriptome and by isolating, culturing and decidualizing EnSCs in vitro. We applied this latter technique for the first time on proliferative endometrial biopsies obtained during ovarian stimulation for in-cycle outcome prediction, in an attempt to overcome inter-cycle variability.
Participants/materials, setting, methods:
RNA-sequencing was performed for 18 individual whole-tissue endometrial biopsies on an Illumina HiSeq1500 machine. DGE was analysed three times using different approaches (DESeq2, EdgeR and the Wilcoxon rank-sum test, all in R). EnSC isolation and IVD was performed (for 2 and 4 days) for a subset of nine samples, after which media from undifferentiated and decidualized cultures were harvested, stored at -80°C and later assayed for 45 cytokines using a multiplex suspension bead immunoassay. The analysis was performed by partial least squares regression modelling.
Main results and the role of chance:
After correction for multiple hypothesis testing, DGE analysis revealed no significant differences between endometrial samples from patients who had a live birth and those with an implantation failure following fresh ET. However secretome analysis after EnSC isolation and culture, showed two distinct clusters that clearly corresponded to the two clinical groups. Upon IVD, the secretome profiles shifted from that of undifferentiated cells but the difference between the two clinical groups remained yet were muted, suggesting convergence of cytokine profiles after decidualization.
Limitations, reasons for caution:
Caution is warranted due to the limited sample size of the study and the in vitro nature of the EnSC experiment. Validation on a larger scale is necessary, however, hard to fulfil given the very limited availability of in-cycle proliferative endometrial biopsies outside a RCT setting.
Wider implications of the findings:
These data support the hypothesis that the endometrium should be assessed not only during the WOI and that certain endometrial dysfunctionalities can probably be detected early in a cycle by making use of the proliferative phase. This insight opens new horizons for the development of endometrial tests, whether diagnostic or predictive of IVF/ICSI treatment outcome.
Study funding/competing interest(s):
This study was supported by Fonds Wetenschappelijk Onderzoek (FWO, Flanders, Belgium, 11M9415N, 1 524 417N), Wetenschappelijk Fonds Willy Gepts (WFWG G160, Universitair Ziekenhuis Brussel, Belgium) and the National Medicine Research Council (NMRC/CG/M003/2017, Singapore). There are no conflicts of interests.
Trial registration number:
NCT02061228.
... It is also necessary for the growth and protection against infection of the spiral arteries in the first month of pregnancy [11,12]. Implantation is stimulated by IFNγ which activates NK cells, regulates colony stimulating factor and promotes the growth of trophoblast [13]. Th1 / Th2 regulatory T cell cytokines are in a delicate balance in pregnancy in order to support fetal growth and to prevent rejection by the maternal immune response (allorejection). ...
... This hypothesis has been verified in our laboratory [26,66,104] in which we found that a difference existed between the role played by the neutrophils and the inflammatory cytokines in both pregnant and non-pregnant cows. According to Hannan and Salamonsen [105], implantation process encompasses a highly regulated temporal and spatial expression of chemokines in the endometrium. This leads not only to specific recruitment and activation of neutrophils but also coordinates proper placentation and angiogenesis. ...
Neutrophils represent the first line of innate immunity and are the most prominent line of cellular defence against invading microorganisms. On stimulation, they can quickly move through the walls of veins and into the tissues of the body to immediately attack or monitor the foreign antigens. Neutrophils are highly versatile and sophisticated cells which are endowed with highly sensitive receptor-based perception systems. They were traditionally classified as short-lived phagocytes actively involved during infection and inflammation, but recently, it has been seen that neutrophils are capable of detecting the presence of sperms during insemination as well as an implanting embryo in the female reproductive tract. These specialised phagocytes play a major role in tissue remodelling and wound healing, and maintain homeostasis during parturition, expulsion of placenta, folliculogenesis, corpus luteum formation and luteolysis. Here, we review the role played by neutrophils in maintaining homeostasis during normal and inflammatory conditions of dairy cattle. We have summarised the alteration in the expression of some cell adhesion molecules and cytokines on bovine neutrophils during different physiological and physiopathological conditions. Some emerging issues in the field of neutrophil biology and the possible strategies to strengthen their activity during the period of immunosuppression have also been discussed.
... Cytokines play an important role in implantation and early pregnancy [16,28]. For example, IL-1 receptors have been shown to be located on the early human embryo, emphasizing the important role of IL1-ra in early pregnancy [18]. ...
Background:
Spontaneous abortion is one of the most common complications in early pregnancy. A preventive test to identify women who will experience a miscarriage, even before first symptoms occur, is not established. Activation of maternal immunological tolerance seems to be essential for early fetal development and various cytokines have been described in different stages of pregnancy. Therefore, we aimed to investigate if chemokine levels at the time of pregnancy testing relative to human Choriogonadotropin (hCG) are altered in patients who will experience a miscarriage in this pregnancy.
Methods:
We obtained blood samples from 39 women. Dependent on the follow-up, patients with a positive pregnancy test were subsequently divided in two groups: ongoing pregnancy (n = 22) and miscarriage (n = 17) in this pregnancy. Immunological and endocrine profiling of maternal plasma at the time of pregnancy testing (5th week of gestation) was performed for each group at the time of pregnancy test using Multiplex and ELISA analysis.
Results:
hCG was significantly decreased in patients with abortion whereas levels of IL-1ra, MIP-1a and TNF-alpha were significantly increased. GCSF/ IL-1ra-ratio was 1.66-fold increased in patients with ongoing pregnancy. TGF-beta /MIP1a-ratio was significantly 3.45-times higher in patients with miscarriage. Comparing patients with ongoing pregnancy to patients experiencing a miscarriage, we could demonstrate significant alterations of the ratios MIP1a/hCG, IL-1ra/hCG, TNFalpha/hCG, MCP1/hCG, IL-6/hCG, TPO/hCG and TGF-beta1/hCG. The strongest effects were seen for the ratio MIP1a/hCG, IL-1ra/hCG and TNFalpha/hCG.
Conclusions:
We have shown that cytokines in relation to hCG after 4 weeks of gestation are significantly altered in women with miscarriage, promising potential as a prognostic biomarker.
... Chemokines were first discovered as mediators of migration of immune cells to sites of inflammation and injury, and now they play multiple roles in organ development, angiogenesis, and tumorigenesis [19]. Chemokines have also been implicated in a number of reproductive events, such as ovulation, menstruation, embryo implantation, parturition and endometriosis [20,21]. Peripheral blood cells are mainly composed of erythrocytes, platelets and leukocytes. . ...
Abstract Background The aim of the present study was to evaluate CCL8 and CXCL10 expression and its regulatory mechanism in peripheral blood leukocytes (PBLs) at the time of maternal recognition in cows. Blood samples were collected on 14, 15, 16, 17 and 18 d after artificial insemination (AI). Based on the day of return of estrus, cows were divided into three groups, pregnant (n = 5), early embryonic mortality (EEM; n = 5) and late embryonic mortality (LEM; n = 5). The gene expression levels in PBLs were assessed with quantitative real-time reverse transcription PCR. Results The expression of CCL8 and CXCL10 mRNA in PBLs gradually increased from 14 to 18 d of pregnant cows and significant differences were observed on 18 d (P
... These factors control trophoblast adhesion and migration, modulate protease activity, and/ or induce cell proliferation and apoptosis. [4][5][6] Chemokines, a large family of chemotactic small molecular weight peptides that contain conserved cysteine motifs in their amino terminus, function to recruit specific leukocytes and activate inflammation. Differential expression and secretion of chemokines induce selective trafficking of leukocyte subsets to the maternal-fetal interface and regulate multiple events that are associated with normal pregnancy. ...
Problem
Certain chemokines with their receptors can promote or inhibit trophoblast cell migration and invasion in human first‐trimester placenta. Whether the lymphotactin (Lptn; XCL1)—XC chemokine receptor 1 (XCR1) chemokine pathway affects trophoblast cell migration and invasion in human first‐trimester placenta remains unclear.
Method of study
The expression pattern of chemokine XCL1 and its receptor XCR1 was detected in human first‐trimester by qRT‐PCR, and the effect of recombinant human XCL1 (rhXCL1) on trophoblast cell function was tested by wound healing and Transwell assays. Matrix metalloproteinase (MMP) activity in trophoblast cells treated with rhXCL1 was assessed via qRT‐PCR and gelatin zymography.
Results
Abundant XCR1 mRNA was expressed in the first‐trimester decidua and villi. XCL1 and XCR1 mRNA were expressed at a higher level in the first‐trimester than in the term placenta. RhXCL1 promoted trophoblast cell migration and invasion by increasing MMP‐9 and MMP‐2 activity and that of the MMP‐2/tissue inhibitor of metalloproteinases 2 (TIMP‐2) complex via the phosphatidylinositol 3‐kinase (PI3K)/AKT kinase (AKT), mitogen‐activated protein kinase (MEK), and JUN N‐terminal kinase (JNK) signaling pathways.
Conclusion
XCL1‐XCR1 chemokine pathway promotes trophoblast invasion by increasing matrix metalloproteinase activity in human first‐trimester placenta.
... In fact, IL-6 levels in amniotic fluid are 20-to 30-fold higher in women with preterm birth associated with infection than in women without infection (Saito et al., 1993). Within the placenta immune-regulatory nature, its capacity of secreting different kinds of chemoattractant molecules, including IL-8, RANTES, and MIP-1α, is not uncommon (Hannan and Salamonsen, 2007). In particular IL-8 is a key chemokine for the activation of neutrophil infiltration that permits adaptive and innate immunity to converge reinforcing the immune response in the placenta (Yuan et al., 2009). ...
Progesterone is an essential hormone that induces deep immune adaptations favoring pregnancy maintenance. We aimed at evaluating the effects of progesterone on the synthesis of pro- and anti-inflammatory cytokines by mononuclear cells isolated from human placental blood stimulated with lipopolysaccharide, emulating an infection-inflammation environment. Mononuclear cells isolated form human placental blood were obtained from nine women undergoing elective cesarean delivery at term (not in labor), isolated by density gradient sedimentation, cultured and co-stimulated with lipopolysaccharide (500 ng/ml) from Escherichia coli in the presence or not of progesterone (0.01, 0.1, or 1.0 µM) for 24 h. Culture supernatants were assayed for pro-inflammatory (IL-1β, TNFα, IL-6), anti-inflammatory (IL-10) cytokines, chemokines (IL-8, MIP-1α) and total MMP-9 by ELISA. In comparison with basal conditions, lipopolysaccharide treatment induced IL-1β, TNFα, IL-6, IL-8, MIP-1α, and MMP-9 synthesis. lipopolysaccharide co-treatment with progesterone significantly decreased the bacterial endotoxin-induced IL-1β, TNF-α, IL-6, IL-8, and MIP-1α secretion. In contrast, co-treatment with progesterone increased the level of IL-10 secreted to the culture medium. The present results support the concept that progesterone can modulate––partially––the inflammatory response of professional immune cells isolated from placental blood. Therefore, progesterone might be part of the natural compensatory mechanism that limits the cytotoxic effects associated with an intrauterine infection process during gestation.
... The number of peer-reviewed articles investigating the genetics of reproductive conditions continues to grow (Fig. 1). Among these published studies is evidence suggesting that subclinical factors, for example dysregulation of ovarian follicle development, hormone metabolism, and immune system function, influence a woman's chances of experiencing reproductive difficulties [3][4][5][6]. Translation of these new findings into genetic testing panels that simultaneously interrogate multiple genetic biomarkers of risk for reproductive conditions is now economically feasible given the decreasing cost of genetic sequencing. ...
... We could not find an appropriate explanation for this apparently contradictory phenomenon. Since chemokine production is regulated by many factors, including cytokines, growth factors, and steroids [17,22,23], other factor(s) may have powerful effects on the stimulation of CCL14 production in the endometrium of early pregnant cows. For example, although IFNT inhibited CCL14 expression in these tissues, FMP showed no effect in this study. ...
The aim of the present study was to determine the possible roles of chemokines in regulating bovine endometrial function during early pregnancy. The expression of six chemokines, including CCL2, CCL8, CCL11, CCL14, CCL16, and CXCL10, was higher in the endometrium at 15 and 18 days of pregnancy than at the same days in non-pregnant animals. Immunohistochemical staining showed that chemokine receptors (CCR1, CCR2, CCR3, and CXCR3) were expressed in the epithelial cells and glandular epithelial cells of the bovine endometrium as well as in the fetal trophoblast obtained from a cow on day 18 of pregnancy. The addition of interferon-τ (IFNT) to an endometrial tissue culture system increased CCL8 and CXCL10 expression in the tissues, but did not affect CCL2, CCL11, and CCL16 expression. CCL14 expression by these tissues was inhibited by IFNT. CCL16, but not other chemokines, clearly stimulated interferon-stimulated gene 15 (ISG15) and myxovirus-resistance gene 1 (MX1) expression in these tissues. Cyclooxygenase 2 (COX2) expression decreased after stimulation with CCL8 and CCL14, and oxytocin receptor (OTR) expression was decreased by CCL2, CCL8, CCL14, and CXCL10. Collectively, the expression of chemokine genes is increased in the endometrium during early pregnancy. These genes may contribute to the regulation of endometrial function by inhibiting COX2 and OTR expression, subsequently decreasing prostaglandin production and preventing luteolysis in cows.
... This also leads to the expression of a unique repertoire of adhesive molecules on the surface of both maternal and fetal cells and thus assists in the attachment of the outer layer of the blastocyst, the trophectoderm, to the endometrial luminal epithelium (Bazer and Johnson 2014). Traditionally recognized for recruiting immune cells to mediate inflammation, chemokines are now known to regulate a range of biological processes including placental development and angiogenesis (Hannan and Salamonsen 2007). Chemokines and their receptors are expressed by a variety of cell types including leukocytes, endothelial cells and trophoblasts suggesting their primary role in physiological or pathological processes during mammalian pregnancy (Du et al. 2014;Griffith et al. 2014). ...
Chemokines play a significant role in pregnancy, especially during embryonic attachment and placental development. During early pregnancy, immune cells are recruited extensively to the endometrium in several species including pigs. However, this recruitment is solely mediated by the presence of the conceptus in pigs making it a unique feature compared with other species (humans, primates and mice). To understand the biological significance of chemokine expression and immune cell recruitment in the context of fetal loss, we investigate a well-characterized porcine fetal loss model during the window of early pregnancy at gestational day (gd) 20 and mid-pregnancy (gd50). These periods coincide with 25–40 % of conceptus loss. Using targeted quantitative polymerase chain reaction and Western blot approaches, we screened a specific set of chemokines. Comparisons were made with endometrial lymphocytes (ENDO LY), endometrium and chorioallantoic membranes (CAM) associated with spontaneously arresting and healthy conceptus attachment sites (CAS). mRNA expression studies revealed an increased expression of CXCR3 and CCR5 in ENDO LY and of CXCL10, CXCR3, CCL5 and CCR5 in the endometrium associated with arresting CAS at gd20. DARC was decreased in the endometrium at gd50. CCL1 was increased in CAM associated with arresting CAS at gd50. Some of these differences were also noted at the protein level (CXCL10, CXCR3, CCL5 and CCR5) in the endometrium and CAM. CD45+ immunohistochemistry demonstrated a significantly higher localization in ENDO LY in the endometrium associated with healthy versus arresting counterparts. Most of these differences were observed in early pregnancy and might contribute towards a shift in immune cell functions.
The actual decrease in the production potential of dairy cattle, particularly due to the reduced reproductive efficiency, is a major concern for the farmers and field veterinarians. Therefore, enhancement of reproductive efficiency is needed to raise the production of dairy cattle. Reproductive failure in terms of infertility, subfertility and dreadful embryonic mortality are some of the major factors responsible for lowering the reproductive efficiency in dairy cattle. Such serious issues can be tackled and minimized by expanding the knowledge in the field of bovine pregnancy and unravel different mechanisms of pregnancy establishment in bovines. This will enable and motivate researchers to discover some possible and suitable interventions around these mechanisms in order to increase the pregnancy outcomes in dairy cattle. Although, there are many mechanisms which have been delineated so far for the pregnancy establishment in bovine but still many areas remain untapped which needs to be taken into limelight. Exploring different mechanisms in early bovine pregnancy may help to improve the reproductive efficiency in bovines and thereby production potential in the dairy sector.
This chapter reviews immune tolerance mechanisms associated with the implantation of the blastocyst and early pregnancy establishment in bovines. During the early pregnancy period, where a majority of embryonic loss occurs, it is imperative for the conceptus not only to prevent the corpus luteal regression but also to modify the maternal immune system from immune rejection. In this regard, interferon tau (IFNτ), a conceptus secreted factor, known to have anti-luteolytic and immunomodulatory properties and is the key pregnancy recognition agent in bovines, plays a preliminary and vital role in the establishment of bovine pregnancy. Whether it is by paracrine or by endocrine manner, IFNτ modulates the maternal immune system and helps in the establishment of pregnancy. Besides IFNτ, another crucial molecule recently came into limelight is indoleamine 2, 3-dioxygenase (IDO), which is an interferon-induced tryptophan catabolic enzyme and is the first and rate-limiting enzyme of the tryptophan catabolism via the kynurenine pathway. It is known to modify the maternal immune system by altering the T cell metabolism and thereby inhibiting the allo-reactive T cell proliferation and at the same time promoting the proliferation of immunosuppressive T regulatory cells. Moreover, to achieve successful implantation, IDO favors an anti-inflammatory cytokine milieu by inducing a shift towards the Th2 cytokines. Although many mechanisms have been delineated so far for the pregnancy establishment in bovine, the focus of this chapter resides on the role of IFNτ and IDO associated Th1-Th2 shift for a successful pregnancy with major emphasis on the tolerogenic mechanism of these molecules.
Pregnancy is intricately regulated by the interactions between various bioactive substances secreted by the conceptus, uterus, and corpus luteum (CL). Interferon-τ, synthesized and secreted by the conceptus, plays a central role in the interaction mechanism of maternal recognition in cows. Chemokines, chemotaxis mediators that are primarily secreted by immune cells, regulate various reproductive responses in various species. Although there are scattered reports on the potential roles of chemokines in the bovine CL and the uterus during the estrous cycle, there is little information on chemokines in these organs during pregnancy. Therefore, in this review, we discuss the possible physiological roles of chemokines in the CL and uterus of pregnant cows, focusing on our recent findings on chemokines and changes in their receptor expression in the CL and endometrium of cows at some stages of pregnancy.
Graphical Abstract Fullsize Image
İmplantasyon; gebelikte embriyo ile endometriyum epiteli arasında sürekli olarak temasın sağlanmasıdır. Endometriyumun implantasyona açık olduğu dönem; implantasyon penceresi olarak tanımlanmaktadır. İmplantasyon penceresi döneminde birçok molekül etkili olmaktadır. Hormonlar, sitokinler, kemokinler, adezyon molekülleri, büyüme faktörleri ve çeşitli genlerin etkisi ile bu süreç koordineli bir şekilde yönetilmektedir. İmplantasyon bu faktörlerin etkisi ile sırasıyla apozisyon, adezyon ve invazyon aşamalarından oluşmaktadır. Bu aşamalar sadece implantasyon penceresinde gerçekleşebilmektedir. Başarılı bir implantasyon olmadan, embriyonun gebeliğin diğer dönemlerine geçişi mümkün değildir ve gebelik erken embriyonik ölümle sonuçlanmaktadır. Bu açıdan multifaktöriyel birçok molekülün koordinasyonuyla meydana gelen implantasyonda, implantasyon penceresi zaman aralığı gebelik sürecindeki kritik noktalardan biridir. Bu derlemede sağlıklı bir gebeliğin oluşabilmesi için gerekli olan başarılı bir implantasyon ve implantasyon penceresi hakkında bilgi verilmeye çalışılmıştır. Fakat bilinmelidir ki, implantasyon mekanizmaları tüm bilinenlere rağmen hala tam olarak aydınlatılamamıştır.
Unlabelled:
Vertebrates have developed various modes of reproduction, some of which are found in Teleosts. Over 300 species of the Syngnathidae (seahorses, pipefishes and seadragons) exhibit male pregnancies; the males have specialized brood pouches that provide immune protection, nourishment, and oxygen regulation. Chemokines play a vital role at the mammalian maternal-fetal interface; however, their functions in fish reproduction are unclear. This study revealed the evolutionary traits and potential functions of chemokine genes in 22 oviparous, ovoviviparous, and viviparous fish species through comparative genomic analyses. Our results showed that chemokine gene copy numbers and evolutionary rates vary among species with different modes of reproduction. Syngnathidae lost cxcl13 and cxcr5, which are involved in key receptor-ligand pairs for lymphoid organ development. Notably, Syngnathidae have site-specific mutations in cxcl12b and ccl44, suggesting immune function during gestation. Moreover, transcriptome analysis revealed that chemokine gene expression varies among Syngnathidae species with different types of brood pouches, suggesting adaptive variations in chemokine functions among seahorses and their relatives. Furthermore, challenge experiments on seahorse brood pouches revealed a joint immune function of chemokine genes during male pregnancy. This study provides insights into the evolutionary diversity of chemokine genes associated with different reproductive modes in fish.
Supplementary information:
The online version contains supplementary material available at 10.1007/s42995-023-00205-x.
Pregnancy is an immunological paradox, with renowned Nobel Prize winning transplantation biologist Sir Peter Brian Medawar being the first to introduce this concept back in 1953. This concept considers how the maternal immune system can tolerate the developing fetus, which is 50% antigenically foreign to the uterus. There have been significant advances in our understanding of the immune system in regulating fertility, pregnancy and in complications of these, and what was once considered a paradox can be seen as a highly evolved system. Indeed, the complexity of the maternal-fetal interface along with our ever-advancing knowledge of immune cells and mediators means that we have a better understanding of these interactions, with gaps still present. This chapter will summarise the key aspects of the role of the immune system at each stage of pregnancy and highlight the recent advances in our knowledge.
The antiphospholipid syndrome (APS) is an autoimmune disease characterized by the persistent presence of antibodies directed against phospholipids and phospholipid-binding proteins that are associated with thrombosis and pregnancy-related morbidity. The latter includes fetal deaths, premature birth and maternal complications. In the early 1990s, a distinct set of autoantibodies, termed collectively antiphospholipid antibodies (aPL), were identified as the causative agents of this disorder. Subsequently histological analyses of the placenta from APS pregnancies revealed various abnormalities, including inflammation at maternal-fetal interface and poor placentation manifested by reduced trophoblast invasion and limited uterine spiral artery remodeling. Further preclinical investigations identified the molecular targets of aPL and the downstream intracellular pathways of key placental cell types. While these discoveries suggest potential therapeutics for this disorder, definitive clinical trials have not been completed. This concise review focuses on the recent developments in the field of basic and translational research pursuing novel mechanisms underlying obstetric APS.
Problem:
In the cell column of anchoring villi, the cytotrophoblast differentiates into extravillous trophoblast (EVT) and invades the endometrium in contact with maternal immune cells. Recently, chemokines were proposed to regulate the decidual immune response. To investigate the roles of chemokines around the anchoring villi, we examined the expression profiles of chemokines in the first-trimester trophoblast-derived Swan71 cells using a three-dimensional culture model.
Method of study:
The gene expressions in the spheroid-formed Swan71 cells were examined by microarray and qPCR analyses. The protein expressions were examined by immunochemical staining. The chemoattractant effects of spheroid-formed Swan71 cells were examined by migration assay using monocyte-derived THP-1 cells.
Results:
The expressions of an EVT marker, laeverin, and matrix metalloproteases, MMP2 and MMP9, were increased in the spheroid-cultured Swan71 cells. Microarray and qPCR analysis revealed that mRNA expressions of various chemokines, CCL2, CCL7, CCL20, CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, and CXCL10, in the spheroid-cultured Swan71 cells were up-regulated as compared with those in the monolayer-cultured Swan71 cells. These expressions were significantly suppressed by hypoxia. Migration assay showed that culture media derived from the spheroid-formed Swan71 cells promoted THP-1 cell migration.
Conclusion:
This study indicated that chemokine expressions in Swan71 cells increase under a spheroid-forming culture and the culture media have chemoattractant effects. Since three-dimensional cell assembling in the spheroid resembles the structure of the cell column, this study also suggests that chemokines play important roles in the interaction between EVT and immune cells in their early differentiation stage.
Fertility of recipient beef cows with subclinical endometritis (SCE) that did or did not receive flunixin meglumine (FM) treatment were compared following transfer of d 7 embryo. The study population comprised of 600 Angus cross cows that expressed estrus following Select-Synch + CIDR (Controlled Internal Drug Release) estrus synchronization protocol. At the time of embryo transfer, approximately 3 wk after sampling for subclinical endometritis, cows were randomly allocated either to receive FM treatment (500 mg of Banamine®; n = 300) or not (Control; n = 300). The effect of subclinical endometritis (at ≥ 1% PMN on endometrial cytology by cytobrush method) and FM treatment on pregnancy/embryo transfer (P/ET, %) were evaluated by mixed model. Of the 600 cows, 323 (53.8%) became pregnant; 55.0% (165/300) cows that received FM treatment vs. 52.7% (158/300) control cows (P > 0.1), and 55.9% (266/476) normal vs. 46.0% (57/124) subclinical endometritis cows (P < 0.05). There was a trend for treatment by subclinical endometritis for P/ET (P = 0.09). Pregnancy was recorded in 55.3% (134/242) of normal and 53.4% (31/58) of subclinical endometritis cows that received FM treatment, and in 56.4% (132/234) of normal and 39.4% (26/66) of subclinical endometritis cows that did not receive FM treatment (P = 0.09). In conclusion, subclinical endometritis in recipient beef cows resulted in lower P/ET. Though not significant in cows with subclinical endometritis, FM treatment resulted in 14.0% points more pregnancy compared with control.
Pregnancy is a complicated physiological process that involves synchronized coordination between immune and endocrine systems. Neutrophils have been suggested as a critical immune cell for embryo implantation and pregnancy maintenance. The present study was conducted to evaluate the dynamic changes in the mRNA expressions of the cluster of designation (CD11b, CD31, CD44 and CD62L) molecules and interferon-stimulated genes (ISG15, MX1 and OAS1) in blood neutrophils throughout pregnancy in dairy cows and correlate them with the outcome of pregnancy. Blood samples were taken from negative control (NC) group, and non-pregnant (NP) group at the time of artificial insemination (AI, day zero) and on days 10, 14, 16, 18, and 21 post-AI. In pregnant (P) cows, samples were taken as described above and after every 30 days until the time of parturition. In aborted cows, samples were collected until the time of the abortion. Comparison between pregnant, non-pregnant and aborted cows revealed that the expression of CD molecules increased (p < 0.05) on days 14, 16, 18 and 21 post-AI only in NP cows as compared to other groups. Although the expression of CD molecules remained constant throughout the study period in pregnant and aborted cows, the expression of CD11b, CD31 and CD62L increased (p < 0.05) on the day of abortion and parturition. Unlike CD molecules, the expression of CD44 decreased significantly (p < 0.05) at the time of abortion. There was a significant (p < 0.05) increase in the expression of interferon-stimulated genes including MX1, OAS1 and ISG15 during the peri-implantation period in pregnant cows, and at the time of abortion in aborted cows. However, the expression of ISGs was lower (p < 0.05) in non-pregnant cows as compared to the other groups. The results revealed the critical role played by neutrophils during pregnancy and form the basis to unravel the underlying mechanism for neutrophil associated immunological infertility in bovines.
Miscarriage can cause significant physical and psychological harm to women. The stromal cell-derived factor 1 (SDF-1, also known as CXCL12)/C-X-C motif chemokine receptor 4 (CXCR4) and C-X-C motif chemokine receptor 7 (CXCR7) axis can promote the proliferation and invasion of trophoblast cells in early pregnancy, and maintain immune tolerance at the maternal-fetal interface to aid with pregnancy success. From our findings, the serum CXCL12 level of women who have miscarried (n = 25) was significantly lower than that of healthy early pregnancy women (n = 20) by ELISA (P < .001). Additionally, CXCL12 levels in normal non-pregnant women (n = 20) were significantly lower than those in early pregnancy women (P < .001) and women who have miscarried (P < .001). Quantitative real-time PCR detected no significant difference in the mRNA transcription levels of CXCR4 and CXCR7 in the decidua tissues of women with early pregnancy (n = 20) and miscarriage (n = 20) (P = .724, P = .281, respectively). However, Western blot and immunohistochemistry of CXCR4 and CXCR7 in decidual tissue of women who have miscarried (n = 20) were significantly lower than those in early pregnancy women (n = 20) (P < .05 for both). Therefore, we believe that the increased serum CXCL12 levels in pregnant offspring may benefit normal pregnancy maintenance. The low level of CXCL12 in peripheral blood and the low expression of CXCR4 and CXCR7 proteins in decidua may be associated with the occurrence of early spontaneous abortion, and the clinical application value of serum CXCL12 in predicting adverse pregnancy outcomes is worth further exploring.
Research question
What is the difference in endometrial transcriptomics between women with normal and with low mid-luteal progesterone during the implantation window?
Design
An endometrial biopsy and serum progesterone concentration were taken from participants during the mid-luteal phase (LH+7 to LH+9). A total of 12 participants were recruited and categorized into two groups based on their progesterone concentrations: normal progesterone (>15 ng/ml, n = 6) and low progesterone (<15 ng/ml, n = 6). Global endometrial gene expression between the two groups was compared by microarray techniques. Principal component analysis was used to display the gene's expression pattern. Pathway and gene ontology enrichment analysis were performed to determine the biological mechanism of progesterone on the endometrium.
Results
Several key genes related to endometrial receptivity were found to be regulated by progesterone. With regard to gene ontology and pathway analysis, progesterone was shown to be mainly involved in structure morphogenesis predominantly during a process of decidualization, extracellular matrix–receptor interaction and cell adhesion. Distinct differences were observed in the transcriptomic profiles between the two groups, indicating potential impairment of endometrial receptivity in women with suboptimal progesterone concentrations. There was a relatively similar pattern of gene expression between endometrial samples with progesterone concentrations approximately 10 ng/ml and >15 ng/ml. Thus, a progesterone concentration of between 10 and 15 ng/ml appears to be sufficient to induce endometrial receptivity.
Conclusions
Abnormally low progesterone below the threshold of 10–15 ng/ml during the implantation window results in aberrant endometrial gene expression that may affect implantation potential.
Among heterogeneous primary tumors of the central nervous system (CNS), gliomas are the most frequent type, with glioblastoma multiforme (GBM) characterized with the worst prognosis. In their development, certain chemokine/receptor axes play important roles and promote proliferation, survival, metastasis, and neoangiogenesis. However, little is known about the significance of atypical receptors for chemokines (ACKRs) in these tumors. The objective of the study was to present the role of chemokines and their conventional and atypical receptors in CNS tumors. Therefore, we performed a thorough search for literature concerning our investigation via the PubMed database. We describe biological functions of chemokines/chemokine receptors from various groups and their significance in carcinogenesis, cancer-related inflammation, neo-angiogenesis, tumor growth, and metastasis. Furthermore, we discuss the role of chemokines in glioma development, with particular regard to their function in the transition from low-grade to high-grade tumors and angiogenic switch. We also depict various chemokine/receptor axes, such as CXCL8-CXCR1/2, CXCL12-CXCR4, CXCL16-CXCR6, CX3CL1-CX3CR1, CCL2-CCR2, and CCL5-CCR5 of special importance in gliomas, as well as atypical chemokine receptors ACKR1-4, CCRL2, and PITPMN3. Additionally, the diagnostic significance and usefulness of the measurement of some chemokines and their receptors in the blood and cerebrospinal fluid (CSF) of glioma patients is also presented.
Atypical chemokine receptor (ACKR) 1, ACKR2, ACKR3, and ACKR4, chemokine decoy receptors that lack G-protein-mediated signaling pathways, internalize and degrade chemokines to control their availability and function. Chemokines play important roles in the endometrium during the estrous cycle and pregnancy, but the expression and regulation of ACKRs have not been determined in pigs. Therefore, we examined the expression of ACKRs in the endometrium throughout the estrous cycle and pregnancy and in conceptus tissues in pigs. ACKR1, ACKR2, ACKR3, and ACKR4 mRNA was expressed in the endometrium, with higher levels of ACKR3 on day 12 of the estrous cycle than in pregnancy and higher levels of ACKR4 on day 15 of pregnancy than in the estrous cycle. ACKR1, ACKR2, and ACKR3, but not ACKR4, mRNA was detected in conceptus and chorioallantoic tissues during pregnancy. ACKR2 and ACKR3 mRNA and ACKR4 protein were mainly localized to luminal epithelial cells and weakly to glandular epithelial cells in the endometrium. Increasing doses of progesterone increased the expression of ACKR2 and ACKR4 and decreased the expression of ACKR3 in endometrial tissues. On day 12 of pregnancy, the expression of ACKR4 mRNA was lower in the endometria of gilts with somatic cell nucleus transfer–derived conceptuses than in the endometria of gilts carrying conceptuses derived from natural mating. These results indicate that the expression of ACKRs is dynamically regulated at the maternal–conceptus interface, suggesting that ACKR proteins might play critical roles in regulating endometrial chemokines to support the establishment and maintenance of pregnancy in pigs. ACKRs are expressed in the porcine endometrium during the estrous cycle and pregnancy and in conceptus tissues.
Purpose:
To investigate the impact of ovarian stimulation and hormone replacement treatment on key regulatory cytokines in endometrial secretion during endometrium implantation window.
Methods:
Fifty-six patients undergoing ovarian stimulation (OS) with gonadotropin releasing hormone antagonist and frozen embryo transfer with hormone replacement treatment (HRT) were recruited. Endometrial secretion aspiration was performed repeatedly during implantation window in natural, OS and HRT cycles of every patient. The concentrations of 17 mediators, known to be involved in human embryo implantation, were assessed by multiplex immunoassay.
Results:
Compared with natural cycle (NC), the concentration of IFN-γ, G-CSF and IL-8 within endometrium were almost the same following OS and HRT. Furthermore, increased MCP-1 levels were observed following HRT and OS. In addition, an increase in IL-1b, IL-7, IL-17, IL-6, TNF-a, IL-12, IL-4, IL-13, IL-10, IL-5, VEGF and MIP-1b concentrations were found in OS cycle only. The level of GM-CSF was lower in HRT cycle and higher in OS cycle, when compared with NC. Among all 17 cytokines, no correlation was found between cytokine concentrations and serum estradiol and progesterone, while only IL-7 concentration has a low correlation with serum LH level.
Conclusion:
Compared to natural and hormone replacement cycle, patients' endometrium cytokine profiles present an increased inflammatory response following ovarian stimulation.
How to Prepare the Endometrium to Maximize Implantation Rates and IVF Success - edited by Gabor Kovacs January 2019
Cambridge Core - Obstetrics and Gynecology, Reproductive Medicine - How to Prepare the Endometrium to Maximize Implantation Rates and IVF Success - edited by Gabor Kovacs
The endometrium develops within the uterus, originating from the urogenital ridge early in development. This mucosal lining consists of epithelial and stromal cells invested with a specialized vasculature that matures at puberty and orchestrates cyclic menses while providing an immunoprotected site for implantation of the fetal allograft. As with other hormone responsive proliferative tissues, such as breast or prostate, the endometrium is prone to pathology. In this chapter we review the physiology of this tissue and explore the mechanisms of the normal and abnormal menstrual cycle. The mechanisms of implantation and menstruation are discussed along with the clinical approach to the evaluation of the endometrium in health and disease.
Objective:
To analyze the transcriptomic profile of endometrial gene alterations during the window of implantation in infertile obese patients.
Design:
Multicenter, prospective, case-control study.
Setting:
Three academic medical centers for reproductive medicine.
Patient(s):
Infertile patients, stratified into body mass index (BMI) categories according to the World Health Organization guidelines, were included in the study.
Intervention(s):
Endometrial samples were obtained from women undergoing standardized estrogen and P replacement cycles after 5 days of vaginal P supplementation.
Main outcome measure(s):
To identify endometrial gene expression alterations that occur during the window of implantation in infertile obese patients as compared with infertile normal-weight controls using a microarray analysis.
Result(s):
XCL1, XCL2, HMHA1, S100A1, KLRC1, COTL1, COL16A1, KRT7, and MFAP5 are significantly dysregulated during the window of implantation in the receptive endometrium of obese patients. COL16A1, COTL1, HMHA1, KRCL1, XCL1, and XCL2 were down-regulated and KRT7, MFAP5, and S100A1 were up-regulated in the endometrium of obese patients. These genes are mainly involved in chemokine, cytokine, and immune system activity and in the structural extracellular matrix and protein-binding molecular functions.
Conclusion(s):
Obesity is associated with significant endometrial transcriptomic differences as compared with non-obese subjects. Altered endometrial gene expression in obese patients may contribute to the lower implantation rates and increased miscarriage rates seen in obese infertile patients.
Clinical trial registration number:
NCT02205866.
Introduction
CXCR2, the receptor of the CXC chemokines, plays a critical role in cell migration and invasion in many types of cancer. It is unclear what impact CXCR2 may have on Preeclampsia (PE), a pregnancy-specific disease, which is related to insufficient trophoblast invasion. The aim of this study was to investigate the expression pattern of CXCR2 in the placentas of healthy and PE pregnancies, and to investigate the molecular mechanism of CXCR2 involvement in the development of PE.
Methods
CXCR2 expression levels in newly delivered placentas from 38 pregnant women with PE and 21 healthy pregnant women were detected using quantitative real-time PCR, immunohistochemistry and Western blot assays. The effect of CXCR2 on trophoblast invasion and the underlying mechanisms were examined in two trophoblast cell lines (HTR-8/SVneo and TEV-1 cells).
Results
CXCR2 mRNA and protein expression levels were significantly decreased in preeclamptic placentas than normal control. The invasive abilities of the two trophoblast cell lines were significantly inhibited when CXCR2 was silenced, but that CXCR2 overexpression promoted trophoblast cells invasion. In addition, silencing CXCR2 reduced the expression of matrix metalloproteinase 2 and 9 (MMP2 and MMP9) and phosphorylated Akt (p-Akt). Furthermore, an Akt inhibitor suppressed the expression of MMP-2 and MMP-9.
Discussion
Our results suggest that the decreased CXCR2 may contribute to the development of preeclampsia through impairing trophoblast invasion by down-regulating MMP-2 and MMP-9 via the Akt signaling pathway.
This chapter summarizes existing information on the synthesis of arylhydrazones of methylene active nitriles (AMANs) and their use as starting materials for the generation of new organic and coordination compounds. Reactions of AMANs with different nucleophiles lead to triazoles, aminopyrazoles, arylazoaminopyrazoles, dicyanopyridazines and other compounds of pharmaceutical significance, depending on the reaction conditions and reagents used. A specific hydrogen bond assisted regioselective activation of a cyano group in a AMAN affords a variety of amidines, carboxamides and iminoesters. Synthesis and structural details of several mono-, tetra- and polynuclear metal complexes of NaI, AgI, NiII, CuII and MnII, III with AMAN ligands are also described. The H-bond and coordination induced E/Z-isomerisation of AMANs can serve for the creation of new molecular switches. Antiferromagnetic interactions are observed in MnII, III-AMAN complexes, while CuII-AMAN complexes are shown to be effective catalyst precursors for the selective oxidation of alcohols to the corresponding carbonyl compounds, and for the diastereoselective nitroaldol (Henry) reaction. Thus, AMANs and their complexes and derivatives can be applied in different fields of modern chemistry, e.g., as pharmaceuticals, molecular magnets, switches and catalyst precursors.
This chapter represents an overview of the existing information on the chemistry and some applications of formazans, compounds with the R1NH-N=CR2-N=NR3 azohydrazone conjugated system. The main method of formazan synthesis involves the interaction of bis-diazo salts with methylene-active and methyne compounds, typical yields being within 10-50%. Representatives of the farmazan family are applied for the synthesis of different heterocycles, while the "formazan - tetrazolium salt" system is widely used as a redox-based staining component for different biochemical studies. Moreover, formazans are valuable spectrofotometric analytical agents and ionophors; they can be used for the simultaneous selective determination of several metal cations. Polymers with formazan moieties are known as selective complexing sorbents for the concentration of micro quantities of elements. The high photo- and thermo-resistance of some formazan complexes allow their application as registering components in optical data storage devices. However, the coordination chemistry of formazans remains essentially undeveloped; only a very limited number of metal complexes with formazan ligands have been fully characterized. Several representatives of formazan complexes are described in this chapter, and some conclusions on the typical coordination modes and other structural details are provided.
The uterus is a site rich in immune cells and is subject to regulation by the sex hormones progesterone and estrogens. Immune modulation within the uterus is initiated during coitus and continues through to the delivery of the baby and for some time postpartum. Several mechanisms to protect the semiallogeneic fetus from maternal immune attack exist, with the carefully regulated recruitment and function of immune cells, as well as the mediators they produce, now recognized as crucial to the success of pregnancy. In the postpartum period, immune cells are also vital to aid the repair and remodeling process, as well as ensure defense against pathogens. Dysregulation of these maternal immune mechanisms can lead to the development of conditions such as preeclampsia, preterm birth, and spontaneous abortion. This chapter will evaluate the immune environment within the uterus and the systemic consequences from coitus to postpartum uterine involution. The effect of pregnancy-associated immune modulation on the symptoms of the autoimmune diseases such as arthritis and systemic lupus erythematosus (SLE) pregnancy-associated infections is also reviewed. © Springer International Publishing Switzerland 2015. All rights are reserved.
The development of a fully functional placenta was crucial to the evolution of human beings. It is the active interface of the most biologically intimate connection between two living organisms: a mother and her fetus. The Evolution of the Human Placenta discusses everything from the organ's methods of protecting the fetus from the mother's own immune system to placental diseases. Starting with some of the earliest events that have constrained or influenced the path of placental evolution in mammals and progressing to the specifics of the human placenta, this book examines modern gestation within an evolutionary framework. Human beings, in terms of evolution, are a successful, rapidly multiplying species. Our reproductive physiology would appear to be functioning quite well. However, human gestation is fraught with many poor outcomes for both the mother and fetus that appear to be-if not unique-far more common in humans than in other mammals. High rates of early pregnancy loss, nausea and vomiting during pregnancy, preeclampsia and related maternal hypertension, and preterm birth are rare or absent in other mammals yet quite typical in humans. Michael L. Power and Jay Schulkin explore more than 100 million years of evolution that led to the human placenta, and in so doing, they help unravel the mysteries of life's earliest moments. © 2012 The Johns Hopkins University Press. All rights reserved.
Endometrial leukocyte subpopulations vary over the reproductive cycle, but no data exist on the mechanism regulating their
recruitment into uterine tissue. This study has evaluated the role of progesterone in the recruitment of selected leukocyte
populations in early pregnancy decidua. Decidua was collected from women in early pregnancy at the time of vacuum aspiration
of the uterus 6, 12, 24 and 36 h after taking 200 mg mifepristone (RU486). Standard immuno-histochemical techniques were employed
to demonstrate the selected leukocyte populations in decidual tissue and these were analysed using image analysis. Fresh decidua
was incubated in medium for 24 h and supernatants assayed for interleukin (IL)-8 (neutrophil chemotactic factor) and MCP-1
(monocyte chemoattractant protein-1) content Analysis of variance demonstrated a significant increase in tissue monocyte number
in decidua 12–36 h after mifepristone administration. No significant changes in other leukocyte subpopulations were observed.
Decidual BL-8 concentrations were significantly increased (P = 0.019) 6 h after mifepristone and decidual MCP-1 concentration rose (non-significant) and fell significantly (P = 0.029) between 6 and 12 h after mifepristone. Progesterone withdrawal may initiate a local cascade of events involving
inflammatory mediators which in turn are responsible for the influx of monocytes. This influx may be essential in the process
of shedding of endometrium or decidua since monocytes and neutrophils are important sources of proteases and collagenases.
Furthermore, these cells are potential local sources of immunomodulatory cytokines.
Endometrial leukocyte subpopulations vary over the reproductive cycle, but no data exist on the mechanism regulating their recruitment into uterine tissue. This study has evaluated the role of progesterone in the recruitment of selected leukocyte populations in early pregnancy decidua. Decidua was collected from women in early pregnancy at the time of vacuum aspiration of the uterus 6, 12, 24 and 36 h after taking 200 mg mifepristone (RU486). Standard immunohistochemical techniques were employed to demonstrate the selected leukocyte populations in decidual tissue and these were analysed using imaged analysis. Fresh decidua was incubated in medium for 24 h and supernatants assayed for interleukin (IL-8) (neutrophil chemotactic factor) and MCP-1 (monocyte chemoattractant protein-1) content. Analysis of variance demonstrated a significant increase in tissue monocyte number in decidua 12-36 h after mifepristone administration. No significant changes in other leukocyte subpopulations were observed. Decidua IL-8 concentrations were significantly increased (P = 0.019) 6 h after mifepristone and decidual MCP-1 concentration rose (non-significant) and fell significantly (P = 0.029) between 6 and 12 h after mifepristone. Progesterone withdrawal may initiate a local cascade of events involving inflammatory mediators which in turn are responsible for the influx of monocytes. This influx may be essential in the process of shedding of endometrium or decidua since monocytes and neutrophils are important sources of proteases and collagenases. Furthermore, these cells are potential local sources of immunomodulatory cytokines.
The endometrium contains a resident population of leukocytes, the number and subtype of which vary throughout the menstrual cycle and in early pregnancy. Factors controlling these fluctuations are unknown, but a combination of proliferation in situ and migration from the vasculature has been proposed. Locally acting inflammatory mediators, including specific chemokines and prostaglandins, have been implicated in these processes. Interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) are potent chemoattractants and activators for neutrophils and monocytes respectively. Locally acting prostaglandins modulate vascular permeability, and a synergistic action of prostaglandin E (PGE) with IL-8 has been described. In the present study IL-8, MCP-1 and cyclooxygenase-2 (COX-2), the inducible isoform of prostaglandin synthase, were all localized in the endometrium by immunohistochemistry throughout the menstrual cycle and in early pregnancy. All three inflammatory mediators were localized to the perivascular cells of blood vessels in endometrium and decidua, and additional immunoreactivity for COX-2 was identified in the glandular epithelium. The intensity of immunostaining was reduced in the periovulatory, early and mid-secretory phases and significantly increased premenstrually. These results further support the hypothesis that there is a premenstrual migration of leukocytes into the endometrium mediated by chemokines.
During the luteal phase and the early months of pregnancy, there is a dense mucosal infiltration of CD56+ natural killer (NK) cells. These uterine NK cells have a phenotype (CD56bright, CD16-, mCD3-) which distinguishes them from peripheral blood NK cells (CD56dim, CD16bright, mCD3-). The uterine NK cells are in close association with extravillous trophoblast (EVT) cells which infiltrate into the decidua and maternal spiral arteries. This subpopulation of trophoblast expresses two human leukocyte antigen (HLA) class I molecules, HLA-G and HLA-C. Circulating NK cells express receptors for HLA class I molecules. We have recently found evidence that similar receptors are present on decidual NK cells belonging to both the Killer Inhibitory Receptor (KIR) and CD94 families. The repertoire of NK receptors expressed varies between different women. The findings indicate that decidual NK cells do have receptors for trophoblast HLA class I molecules. Experiments are underway to determine the effects of this interaction on NK cell function.
Chemokines are a family of small polypeptides which specialize in the attraction of leukocytes. The presence of specific leukocyte subsets at the implantation site is an important element of the complex, and not completely understood, process of embryonic implantation. This report includes the investigation of the in-vivo immunolocalization and hormonal regulation of interleukin (IL)-8, monocyte chemotactic protein (MCP)-1 and RANTES (regulated upon activation normal T-cell expressed and secreted) in the human endometrium during hormone replacement therapy cycles for oocyte recipients in an IVF programme. In addition, we have analysed the embryonic regulation of these endometrial epithelial chemokines (IL-8 and MCP-1) using an in-vitro model for the apposition phase of human implantation by co-culturing single human embryos until the blastocyst stage with human endometrial epithelial cells (EEC). IL-8 and MCP-1 were immunolocalized in the human endometrium to the glandular and lumenal epithelium as well as to the endothelial cells. RANTES was mainly localized to the stromal compartment and endothelial cells. The immunoreactive levels of endometrial IL-8 and MCP-1 were up-regulated by the administration of progesterone during the receptive phase of the cycle. Furthermore, it was demonstrated that, in vitro, the human blastocyst does not produce measurable amounts of IL-8, MCP-1 or RANTES; however, it does up-regulate EEC IL-8 mRNA expression (P < 0.05) and protein production (P < 0.05), but not IL-8 secretion. The human embryo did not regulate EEC MCP-1 expression. These results provide evidence of hormonal and embryonic regulation of specific endometrial chemokines, suggesting two different but related mechanisms to induce the production of chemokines by the EEC, thus contributing to the attraction of specific leukocyte populations during the peri-implantation phase.
Trophoblast adhesion to the uterine wall is the requisite first step of implantation and, subsequently, placentation. At the
maternal-fetal interface, we investigated the expression of selectin adhesion systems that enable leukocyte capture from the
bloodstream. On the maternal side, human uterine epithelial cells up-regulated selectin oligosaccharide-based ligands during
the window of receptivity. On the fetal side, human trophoblasts expressed L-selectin. This ligand-receptor system was functional,
because beads coated with the selectin ligand 6-sulfo sLex bound to trophoblasts, and trophoblasts bound to ligand-expressing uterine luminal epithelium in tissue sections. These results
suggest that trophoblast L-selectin mediates interactions with the uterus and that this adhesion mechanism may be critical
to establishing human pregnancy.
At the human feto-maternal interface, trophoblasts differentiate towards extravillous trophoblasts (EVTs) and form the cell column. EVTs acquire invasive activity in the distal part of the cell column and begin to migrate into the maternal tissue. We previously reported that dipeptidyl peptidase IV(DPPIV) is expressed on EVTs in the proximal part of cell column and is involved in the inhibition of their migration. Because DPPIV has been shown to degrade several chemokines, we examined possible roles of chemokines in EVT migration.
Immunohistochemistry demonstrated that C-C chemokine receptor 1 (CCR1) was hardly detected on cytotrophoblasts and syncytiotrophoblast but was expressed on EVTs in the cell column. In vitro, CCR1 protein was also present on the surface of EVTs that grew out from chorionic villous explants cultured under 20% O2. Chemokines that can bind to CCR1 (CCR1 ligands), such as regulated on activation, normal T cell expressed and secreted (RANTES) and macrophage inflammatory protein-1α (MIP-1α), were confirmed in the decidual tissues by RT-PCR and immunohistochemistry. These CCR1 ligands promoted the migration of the EVTs that were isolated from the explant cultures in vitro. These results indicate that CCR1 is expressed on trophoblasts as they differentiate to EVTs and that CCR1 ligands produced from the decidual tissue induce EVT migration.
By contrast, CCR1 was scarcely expressed on EVTs that grew out from villous explants cultured in 1% O2, indicating that a relatively high oxygenic environment is needed to induce CCR1 expression. Moreover, CCR1 expression on the isolated EVTs was significantly reduced in the presence of decidua-conditioned medium. Such regulation of CCR1 by surrounding oxygenic and decidual environments supports a close correlation between EVT invasion and their expression of CCR1.
This study demonstrates that trophoblasts acquire CCR1 as they differentiate to an invasive phenotype at the villus-anchoring sites and indicates a novel role for the chemokine-CCR1 system in the initial step of trophoblastic invasion towards the maternal tissue.
In this study, we address the question of the cross-talk between two chemokines that are cosecreted during inflammation, namely monocyte chemoattractant protein-1 (MCP-1) and soluble fractalkine (s-FKN), toward monocyte migration. We found that s-FKN fails to induce MonoMac6 cell migration per se. Interestingly, this chemokine antagonizes transendothelial migration and chemotaxis of MonoMac6 cells and freshly isolated human monocytes induced by MCP-1, indicating a direct effect of s-FKN on monocytic cells. In this study, we found that stress-activated protein kinase (SAPK)1/c-Jun N-terminal kinase 1 and SAPK2/p38 are involved in the control of MCP-1-induced MonoMac6 cell migration. We demonstrated that s-FKN abrogates the MCP-1-induced SAPK2/p38 activation as well as the upstream Pyk2 activity. Furthermore, we observed that s-FKN also inhibits the activity of a major matrix metalloproteinase (MMP), namely MMP-2. Taken collectively, our results indicate that the s-FKN antagonizes the chemoattractant effect of MCP-1 on monocytes, likely by inhibiting crucial signaling pathways, like SAPK2/p38 and MMP-2 activities.
In this study, we investigated the expression of ligands for CXCR3 (Mig, IP-10, and I-TAC) and CXCR4 (SDF-1) in the human endometrium throughout the menstrual cycle. By immunohistochemistry, immunostaining for Mig and IP-10 was found in the surface epithelia, glandular epithelia, and stroma with some menstrual cycle-dependent fluctuation. By contrast, immunostaining for I-TAC or SDF-1 was not detected. ELISA demonstrated that the concentrations of Mig and IP-10 were higher in the secretory phase than in the proliferative phase, but I-TAC and SDF-1alpha was detected in only a few samples. Endometrial Mig and IP-10 concentrations strongly correlated with the number of endometrial natural killer cells. Progesterone significantly induced Mig secretion and tended to induce IP-10 secretion from the cultured endometrial stromal cells, whereas 17beta-estradiol had no significant effect. Neither I-TAC nor SDF-1alpha was detected in the supernatant of cultured endometrial stromal cells in the presence or absence of 17beta-estradiol or progesterone. The results suggest that Mig and IP-10 may be involved in the recruitment of natural killer cells or other phenomena in the human endometrium.
Human endometrium possesses a unique immunological environment enabling implantation of the semiallogeneic embryo. Large populations of macrophages and uterine-specific natural killer cells infiltrate the implantation site, believed to be important modulators of trophoblast invasion and decidualization. In the absence of pregnancy, there is a dramatic influx of neutrophils, eosinophils, and macrophages, likely to be critical for focal inflammatory endometrial destruction. However, little is known regarding selective recruitment of leukocyte subtypes. We employed a gene array approach to analyze the expression of 21 chemokines in endometrium. Real-time RT-PCR and immunohistochemistry was conducted to verify expression patterns and determine cellular source. Nine chemokines were highly abundant in human endometrium: monocyte chemotactic protein-3, eotaxin, fractalkine, macrophage inflammatory protein-1beta, 6Ckine, IL-8, hemofiltrate CC chemokine-1 and -4, and macrophage-derived chemokine. Chemokine mRNA was generally up-regulated during endometrial receptivity and early pregnancy, particularly of macrophage and natural killer chemoattractants. Chemokine protein was predominantly localized to epithelial glands, whereas differentiated stromal cells were a major source of chemokines after decidualization. This is the first study to use an unbiased approach to screen for endometrial chemokines, and we report the selective regulation of chemokines, corresponding to the recruitment of distinct leukocyte subpopulations required for pregnancy and menstruation.
Macrophage inflammatory protein-1alpha (MIP-1alpha) is a chemokine that leads to leukocyte recruitment and activation at sites of infection. Controlling chemokine activity at sites of infection is important, since excess accumulation of leukocytes may contribute to localized tissue damage. Neutrophil-derived serine proteases modulate the bioactivity of chemokine and cytokine networks through proteolytic cleavage. Because MIP-1alpha is temporally expressed with neutrophils at sites of infection, we examined proteolysis of MIP-1alpha in vitro by the neutrophil-derived serine proteases: cathepsin G, elastase, and proteinase 3. Recombinant human MIP-1alpha isoforms LD78beta and LD78alpha were expressed and purified, and the protease cleavage sites were analyzed by mass spectrometry and peptide sequencing. Chemotactic activities of parent and cleavage molecules were also compared. Both LD78beta and LD78alpha were cleaved by neutrophil lysates at Thr16-Ser17, Phe24-Ile25, Tyr28-Phe29, and Thr31-Ser32. This degradation was inhibited by serine protease inhibitors phenylmethylsulfonyl fluoride and 4-(2-aminoethyl)-benzenesulfonyl fluoride. Incubation of the substrates with individual proteases revealed that cathepsin G preferentially cleaved at Phe24-Ile25 and Tyr28-Phe29, whereas elastase and proteinase 3 cleaved at Thr16-Ser17 and Thr31-Ser32. Proteolysis of LD78beta resulted in loss of chemotactic activity. The role of these proteases in LD78beta and LD78alpha degradation was confirmed by incubation with neutrophil lysates from Papillon-Lefevre syndrome patients, demonstrating that the cell lysates containing inactivated serine proteases could not degrade LD78beta and LD78alpha. These findings suggest that severe periodontal tissue destruction in Papillon-Lefevre syndrome may be related to excess accumulation of LD78beta and LD78alpha and dysregulation of the microbial-induced inflammatory response in the periodontium.
Chemokines are implicated in the implantation process. The aim of this study was to investigate mRNA expression and protein levels of chemokine receptors CXCR1, CXCR4, CCR5 and CCR2B in human endometrium throughout the menstrual cycle, during HRT and in the human blastocyst. The regulation of chemokine receptors in the endometrial epithelium was also studied using an in-vitro model for the apposition phase of human implantation. We found up-regulation of endometrial CXCR1 mRNA (419-fold increase), CCR5 mRNA (612-fold increase) and CCR2B mRNA (657 fold-increase) during the luteal phase peaking in the pre-menstrual endometrium. CXCR4 mRNA levels presented a specific although modest (18-fold increase) up-regulation during the implantation window. These findings were corroborated at the protein level in natural and HRT cycles. Immunoreactive CCR5 and CCR2B receptors were detected in human blastocysts whereas CXCR4 and CXCR1 were not present. Chemokine receptors in cultured endometrial epithelial cells showed an up-regulation and polarization of CXCR1, CXCR4 and CCR5 receptors when a human blastocyst was present. The specific distribution and regulation of chemokine receptors in the endometrial epithelium and the human blastocyst suggest a possible implication of these receptors in the apposition and adhesion phases of human implantation.
More than 70% of decidual lymphocytes are NK cells characterized by CD56(bright)CD16(-) phenotype, but the mechanisms by which these NK cells are recruited in the decidua are still almost unrevealed. In this study, we first analyzed the transcription of 18 chemokine receptors in the first-trimester decidual CD56(bright)CD16(-) NK cells. Among these receptors, CXCR4 and CXCR3 were found highly transcribed, and the expression of CXCR4 was verified in most of the decidual CD56(bright)CD16(-) NK cells by flow cytometry. The first-trimester human trophoblasts were found expressing CXCL12/stromal cell-derived factor 1, the specific ligand of CXCR4, by way of in situ hybridization and immunohistochemistry. The primary cultured trophoblast cells were also found to secrete stromal cell-derived factor 1alpha spontaneously, and its concentration was 384.6 +/- 90.7 pg/ml after the trophoblast cells had been cultured for 60 h. All of the ligands for CXCR3 were below the minimal detectable concentration when trophoblast cells were cultured for up to 48 h. Both recombinant human SDF-1alpha and supernatants of the cultured trophoblast cells exhibited chemotactic activity on decidual CD56(bright)CD16(-) NK cells. Our findings suggest that human first-trimester trophoblast cells produce CXCL12, which in turn chemoattracts decidual CD56(bright)CD16(-) NK cells. This activity could contribute to the recruitment mechanism of decidual lymphocytes, especially CD56(bright)CD16(-) NK cells, in decidua, and may be used at a local level to modulate the immune milieu at the materno-fetal interface.
A clear parallelism between the different steps in human embryo-endometrial apposition/adhesion/invasion and leukocyte-endothelium rolling/adhesion/extravasation can be established. During human implantation and leukocyte trafficking, a first wave of soluble mediators regulates the expression and functional activity of adhesion molecules such as L-selectin and integrins, which mediate both processes. Apical surfaces of human endometrial epithelium and endothelium are key elements for the initiation of molecular interactions to capture the blastocyst or leukocyte, respectively. Subsequently, the blastocyst and the leukocyte migrate through the epithelium and endothelium toward their final destination, the endometrial stroma, to initiate placentation or the inflammatory foci as part of the immune response. Similarities between the intermediate molecular mechanisms of these two physiologically unrelated processes are discussed.
The complexity of the events of embryo implantation and placentation is exemplified by the number and range of cytokines with
demonstrated roles in these processes. Disturbance of the normal expression or action of these cytokines results in complete
or partial failure of implantation and abnormal placental formation in mice or humans. Of known importance are members of
the gp130 family such as interleukin-11 (IL-11) and leukaemia inhibitory factor (LIF), the transforming growth factor β (TGFβ)
superfamily including the activins, the colony-stimulating factors (CSF), the IL-1 system and IL-15 system. New data are also
emerging for roles for a number of chemokines (chemoattractive cytokines) both in recruiting specific cohorts of leukocytes
to implantation sites and in trophoblast differentiation and trafficking. This review focuses on those cytokines and chemokines
whose expression pattern in the human endometrium is consistent with a potential role in implantation and placentation and
for which some relevant actions are known. It examines what is known of their regulation and action along with alterations
in clinically relevant situations.
In humans, fetal cytotrophoblasts leave the placenta and enter the uterine wall, where they preferentially remodel arterioles. The fundamental mechanisms that govern these processes are largely unknown. Previously, we have shown that invasive cytotrophoblasts express several chemokines, as well as the receptors with which they interact. Here, we report that these ligand-receptor interactions stimulate cytotrophoblast migration to approximately the same level as a growth factor cocktail that includes serum. Additionally, cytotrophoblast commitment to uterine invasion was accompanied by rapid downregulation of EPHB4, a transmembrane receptor associated with venous identity, and upregulation of ephrin B1. Within the uterine wall, the cells also upregulated expression of ephrin B2, an EPH transmembrane ligand that is associated with arterial identity. In vitro cytotrophoblasts avoided EPHB4-coated substrates; upon co-culture with 3T3 cells expressing this molecule, their migration was significantly inhibited. As to the mechanisms involved, cytotrophoblast interactions with EPHB4 downregulated chemokine-induced but not growth factor-stimulated migration. We propose that EPHB4/ephrin B1 interactions generate repulsive signals that direct cytotrophoblast invasion toward the uterus, where chemokines stimulate cytotrophoblast migration through the decidua. When cytotrophoblasts encounter EPHB4 expressed by venous endothelium, ephrin B-generated repulsive signals and a reduction in chemokine-mediated responses limit their interaction with veins. When they encounter ephrin B2 ligands expressed in uterine arterioles, migration is permitted. The net effect is preferential cytotrophoblast remodeling of arterioles, a hallmark of human placentation.
Successful implantation relies on the tightly regulated invasion of extravillous trophoblasts (EVTs). However, little is known about their phenotypic differentiation and relevant motile behaviour. Furthermore, the cell-cell interactions between EVTs and decidual arterioles during physiological transformation are also poorly understood.
A total of 128 decidual specimens from early and late gestations containing components of EVTs and spiral arterioles were investigated using immunohistochemistry and periodic acid-Schiff reaction.
Unipolar, tadpole-like EVTs are observed throughout the interstitial area, with a tendency to decrease along the invasive pathway. The stellate differentiation of the EVTs is identified around and inside decidual arterioles or in the third-trimester myometrium. Furthermore, stellate transformation of EVTs precedes its interactions with the decidual arteriole. These specialized stellate trophoblasts invade and infiltrate the tunica media, accompanying lacuna formation inside the vessel wall and perturbation of actin fibre alignment of the tunica media.
Stellate transformation of trophoblasts may explain controlled invasion of EVTs and probably plays a key role in initiating cell-cell interaction in decidual vascular remodelling.
Human CD56(bright) NK cells accumulate in the maternal decidua during pregnancy and are found in direct contact with fetal trophoblasts. Several mechanisms have been proposed to explain the inability of NK cells to kill the semiallogeneic fetal cells. However, the actual functions of decidual NK (dNK) cells during pregnancy are mostly unknown. Here we show that dNK cells, but not peripheral blood-derived NK subsets, regulate trophoblast invasion both in vitro and in vivo by production of the interleukin-8 and interferon-inducible protein-10 chemokines. Furthermore, dNK cells are potent secretors of an array of angiogenic factors and induce vascular growth in the decidua. Notably, such functions are regulated by specific interactions between dNK-activating and dNK-inhibitory receptors and their ligands, uniquely expressed at the fetal-maternal interface. The overall results support a 'peaceful' model for reproductive immunology, in which elements of innate immunity have been incorporated in a constructive manner to support reproductive tissue development.
During the invasive phase of implantation, trophoblasts and maternal decidual stromal cells secrete products that regulate trophoblast differentiation and migration into the maternal endometrium. Paracrine interactions between the extravillous trophoblast and the maternal decidua are important for successful embryonic implantation, including establishing the placental vasculature, anchoring the placenta to the uterine wall, and promoting the immunoacceptance of the fetal allograph. To our knowledge, global crosstalk between the trophoblast and the decidua has not been elucidated to date, and the present study used a functional genomics approach to investigate these paracrine interactions. Human endometrial stromal cells were decidualized with progesterone and further treated with conditioned media from human trophoblasts (TCM) or, as a control, with control conditioned media (CCM) from nondecidualized stromal cells for 0, 3, and 12 h. Total RNA was isolated and processed for analysis on whole-genome, high-density oligonucleotide arrays containing 54,600 genes. We found that 1374 genes were significantly upregulated and that 3443 genes were significantly downregulated after 12 h of coincubation of stromal cells with TCM, compared to CCM. Among the most upregulated genes were the chemokines CXCL1 (GRO1) and IL8,CXCR4, and other genes involved in the immune response (CCL8 [SCYA8], pentraxin 3 (PTX3), IL6, and interferon-regulated and -related genes) as well as TNFAIP6 (tumor necrosis factor alpha-induced protein 6) and metalloproteinases (MMP1, MMP10, and MMP14). Among the downregulated genes were growth factors, e.g., IGF1, FGF1, TGFB1, and angiopoietin-1, and genes involved in Wnt signaling (WNT4 and FZD). Real-time RT-PCR and ELISAs, as well as immunohistochemical analysis of human placental bed specimens, confirmed these data for representative genes of both up- and downregulated groups. The data demonstrate a significant induction of proinflammatory cytokines and chemokines, as well as angiogenic/static factors in decidualized endometrial stromal cells in response to trophoblast-secreted products. The data suggest that the trophoblast acts to alter the local immune environment of the decidua to facilitate the process of implantation and ensure an enriched cytokine/chemokine environment while limiting the mitotic activity of the stromal cells during the invasive phase of implantation.
Chemokines are a family of small polypeptides which specialize in the attraction of leukocytes. The presence of specific leukocyte subsets at the implantation site is an important element of the complex, and not completely understood, process of embryonic implantation. This report includes the investigation of the in-vivo immunolocalization and hormonal regulation of interleukin (IL)-8, monocyte chemotactic protein (MCP)-1 and RANTES (regulated upon activation normal T-cell expressed and secreted) in the human endometrium during hormone replacement therapy cycles for oocyte recipients in an IVF programme. In addition, we have analysed the embryonic regulation of these endometrial epithelial chemokines (IL-8 and MCP-1) using an in-vitro model for the apposition phase of human implantation by co-culturing single human embryos until the blastocyst stage with human endometrial epithelial cells (EEC). IL-8 and MCP-1 were immunolocalized in the human endometrium to the glandular and lumenal epithelium as well as to the endothelial cells. RANTES was mainly localized to the stromal compartment and endothelial cells. The immunoreactive levels of endometrial IL-8 and MCP-1 were up-regulated by the administration of progesterone during the receptive phase of the cycle. Furthermore, it was demonstrated that, in vitro, the human blastocyst does not produce measurable amounts of IL-8, MCP-1 or RANTES; however, it does up-regulate EEC IL-8 mRNA expression (P < 0.05) and protein production (P < 0.05), but not IL-8 secretion. The human embryo did not regulate EEC MCP-1 expression. These results provide evidence of hormonal and embryonic regulation of specific endometrial chemokines, suggesting two different but related mechanisms to induce the production of chemokines by the EEC, thus contributing to the attraction of specific leukocyte populations during the peri-implantation phase.
Matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes, capable of degrading proteins found in the extracellular matrix. MMPs 2 and 9 are known to be produced by microglia, the resident macrophages of the central nervous system. The control of the secretion of these proteases and the activation of proenzymes by other proteases such as plasmin, as well as the balance between MMP secretion and the secretion of their natural inhibitors (TIMPs), have an important relevance in the pathogenesis of multiple sclerosis (MS). The in vitro control of MMPs 2 and 9, TIMPs 1 and 2, and urokinase-type plasminogen activator by microglia was examined in response to a panel of chemokines (chemotactic cytokines), using ELISA and zymography techniques. The chemokines MCP1, MIP1β, RANTES, IL-8, and Fractalkine were all found significantly to increase the secretion of MMPs and TIMPs by a human foetal microglial cell line, CHME3, after 24 h stimulation. The chemokines tested, MCP1, MIP1β, and Fractalkine, were also shown to increase MMP9 secretion by primary isolated rat brain microglia in vitro. MCP1, MIP1α/β, and RANTES significantly decreased the secretion of uPA into culture supernatants in ELISA experiments. These findings suggest an important potential role for the involvement of chemokines in the breakdown of the blood–brain barrier and also the destruction of myelin basic protein in MS. GLIA 28:183–189, 1999. © 1999 Wiley-Liss, Inc.
Objective To investigate the distribution and the role of macrophage inflammatory protein-la (MIP- 1α) in human endometrium during cyclic changes.
Setting Department of Obstetrics and Gynaecology, Shiga University of Medical Science and University Hospital.
Materials Eighteen endometrial tissue specimens surgically resected or biopsied from women with normal menstrual cycles, without hormonal disorder or endometrial diseases.
Methods By immunohistochemistry, using monoclonalantibodies (lambda delta 78 for MIP-la and CR3/43 for human leukocyte antigen-DR (HLA-DR)).
Results Immunoreactivity for anti-MIP-lα was distributed diffusely in epithelial cells throughout the proliferative and secretory phases but was absent during menstruation due to degenerative or necrotic changes. HLA-DR was expressed in epithelial cells only in the late secretory phase and was not expressed in stromal cells.
Conclusion Immunohistochemicalanalysis showed the presence of MIP- lα in the endometrial epithelium. Expression of HLA-DR in epithelial cells was observed only in the late secretory phase, suggesting that accumulation of MIP-lα in epithelium occurred by self production and not via a receptor mediated pathway. MIP-lα was released from the denuded epithelium during menstruation and appeared to contribute to the accumulation of monocytes/macrophages into the endometrial cavity. MIP-la has a number of biological effects other than monocytic chemotaxis, and some of these effects may be exerted in the endometrial tissue.
Menstruation occurs at the end of a normal reproductive cycle in the human female, following the fall in progesterone resulting from the demise of the corpus luteum. Current data support a central role for the matrix metalloproteinases in menstruation but their focal pattern of expression within peri-menstrual and menstrual endometrium suggests local rather than hormonal regulation. This review emphasizes the similarities between menstruation and an inflammatory process and examines the relationship between cells of hemopoietic lineage, particularly mast cells, eosinophils, neutrophils and macrophages, and the local production and activation of matrix metalloproteinases within the endometrium. It proposes a complex of critical regulatory circuits, initially activated by the withdrawal of progesterone, which provide interactions between the migratory cells that produce a myriad of important regulatory molecules and endometrial stromal and epithelial cells which produce both chemokines and matrix metalloproteinases. These mechanisms could account for the focal nature of the tissue degradation at menstruation.
During human pregnancy, the uterus is infiltrated by a population of maternal leukocytes that co-exist with fetal cytotrophoblasts occupying the decidua and uterine blood vessels. These immune cells, termed “decidual granulated leukocytes,” are composed predominately (70%) of the CD56bright subset of natural killer cells, accompanied by T cells (15%) and macrophages (15%). The mechanisms underlying the recruitment of these cells are unknown, but by analogy to other systems, chemokines are likely to be involved. We examined the expression patterns of 14 chemokines in the decidualized uterine wall by in situ hybridization, and the expression of chemokine receptors on decidual leukocytes by RNase protection. The striking concordance between the expression of chemokines in the uterus and their receptors on decidual leukocytes allowed us to identify numerous receptor-ligand pairs that may recruit the latter cells to the uterus during pregnancy. Additionally, chemokine expression patterns suggested other, nonimmune functions for these molecules, including a role in cytotrophoblast differentiation. Together, our results imply that chemokine networks serve important functions at the maternal-fetal interface.
Development of the human embryo depends on the ability of first trimester cytotrophoblastic stem cells to differentiate and invade the uterus. In this process, transient expression of an invasive phenotype is part of normal cytotrophoblast differentiation. Morphologically, this process begins when polarized chorionic villus cytotrophoblasts form multilayered columns of nonpolarized cells, and invade the uterus. Using immunocytochemistry, we compared the presence of adhesion receptors and extracellular matrix ligands on cytotrophoblasts in villi, cell columns, and the uterine wall. Villus cytotrophoblasts, anchored to basement membrane, stained for alpha 6 and beta 4 integrin subunits and both merosin and A-chain-containing laminin. Nonpolarized cytotrophoblasts in columns expressed primarily alpha 5 and beta 1 integrin subunits and a fibronectin-rich matrix. Cytotrophoblast clusters in the uterine wall stained for alpha 1, alpha 5, and beta 1 integrins, but not for most extracellular matrix antigens, suggesting that they interact primarily with maternal cells and matrices. Tenascin staining was restricted to stroma at sites of transition in cytotrophoblast morphology, suggesting that tenascin influences cytotrophoblast differentiation. Our results suggest that regulation of adhesion molecule expression contributes to acquisition of an invasive phenotype by cytotrophoblasts and provide a foundation for studying pathological conditions in which insufficient or excessive trophoblast invasion occurs, such as preeclampsia or choriocarcinoma.
Trophoblastic invasion of the human decidua has been studied in 48 intact uteri with pregnancies ranging from 8 to 18 weeks after the last menstrual period. Some cytotrophoblast invades the distal segments of the spiral arteries to become endovascular while the rest diffusely infiltrates the decidua as an interstitial invader. The interstitial cytotrophoblast reaches the myometrium and gives rise to the characteristic placental bed giant cells. As the placental site enlarges the lateral spiral arteries come to lie obliquely; new openings into the intervillous space are created but this readjustment of the placental blood supply may cause focal superficial decidual necrosis. The physiological changes converting the spiral to the uteroplacental arteries are effected in the upper decidua by the action of endovascular and perivascular cytotrophoblast, whereas in the deeper decidua endovascular trophoblast is principally involved. Endometrial granulocytes aggregate in the region of maternal tissue degeneration with the heaviest trophoblast invasion but the role played by these cells in placentation is unknown.
The cytokine interleukin-8 (IL-8) is capable of inducing selective neutrophil chemotaxis and activation and has been postulated as the signal for neutrophil recruitment and activation in reproductive tissues. The aim of this study was to examine the localization of IL-8 in non-pregnant human endometrium in view of its potential as a local modulator of endometrial function. Endometrial biopsies (proliferative, n = 8; secretory, n = 7) from 15 subjects were available for immunolocalization of IL-8. Primary antibody (rabbit polyclonal against IL-8) application was followed by either a horseradish peroxidase streptavidin detection system (proliferative, n = 5; secretory, n = 4) or an avidin-biotin alkaline phosphatase detection method (proliferative, n = 3; secretory, n = 3). All endometrial biopsies showed heterogeneous positive staining for IL-8 in association with blood vessels in both proliferative and secretory phase biopsies. The immunostaining was apparently not associated with endothelial cells but rather appeared to be associated with the smooth muscle layer of arterioles. Secretory phase biopsies exhibited immunoreactivity in association with small blood vessels and spiral arterioles. The perivascular location of IL-8 throughout the stages of the human menstrual cycle is consistent with its proposed biological role as a modulator of endometrial function, especially synergism with prostaglandin E and the transmigration of leukocytes.
Chemokines are a new family of cytokines specialised in attracting leukocytes, acting in physiological conditions and in pathological processes. A wide variety of cell types in response to exogenous irritants or endogenous mediators of the inflammatory reaction produce them. Pivotal parts of reproductive function are based on inflammatory like processes wherein different leukocytes subsets are recruited and activated to produce paracrine autocrine effects in which cytokines and growth factors are implicated. Since chemokines control leukocyte trafficking and belong to the cytokine superfamily, in this review we analyze the implications of these molecules and related cells in ovulation, embryonic implantation, menstruation, parturition and their role in pathological process such as preterm delivery, endometriosis, ovarian hyperstimulation syndrome and HIV infection.
To review the available information regarding chemotactic cytokines and their possible implications in human reproduction.
A thorough literature and MEDLINE search was conducted to identify studies relating to the role of chemokines in ovulation, menstruation, implantation, cervical ripening and preterm labor, and endometriosis.
Chemokines mediate leukocyte traffic through their specific receptors in various tissues. Although four families have been described to date, two remain the major subfamilies: alpha-chemokines (with interleukin-8 as representative for this group), and beta-chemokines (with monocyte chemotactic protein-1 as representative). Interleukin-8, monocyte chemotactic protein-1, and growth-regulated oncogene-alpha are involved in follicular development and atresia, ovulation, steroidogenesis, and corpus luteum function. Interleukin-8 showed cycle-dependent expression in human endometrium, and at the same time, stimulated endometrial stromal cell growth, acting as an autocrine growth factor. Interleukin-8 has been identified in human amnion, chorion, decidua, and villous placenta, and its level increases during labor. Levels of interleukin-8 correlate with the release of collagenases, a crucial step that regulates the process of cervical extracellular matrix remodeling. The levels of monocyte chemotactic protein-1; regulated on activation, normal T-expressed and secreted (RANTES); interleukin-8; and growth-regulated oncogene-alpha are elevated in the peritoneal fluid of women with endometriosis, and they correlate with the stage of the disease.
Chemokines play a relevant role in many physiologic and pathologic situations, such as ovulation, menstruation, implantation, cervical ripening and preterm labor, and endometriosis. Their regulation soon may provide new therapeutic strategies.
To investigate the distribution and the role of macrophage inflammatory protein-1alpha (MIP-1alpha) in human endometrium during cyclic changes.
Department of Obstetrics and Gynaecology, Shiga University of Medical Science and University Hospital.
Eighteen endometrial tissue specimens surgically resected or biopsied from women with normal menstrual cycles, without hormonal disorder or endometrial diseases.
By immunohistochemistry, using monoclonal antibodies (lambda delta 78 for MIP-1alpha and CR3/43 for human leukocyte antigen-DR (HLA-DR)).
Immunoreactivity for anti-MIP-1alpha was distributed diffusely in epithelial cells throughout the proliferative and secretory phases but was absent during menstruation due to degenerative or necrotic changes. HLA-DR was expressed in epithelial cells only in the late secretory phase and was not expressed in stromal cells.
Immunohistochemical analysis showed the presence of MIP-1alpha in the endometrial epithelium. Expression of HLA-DR in epithelial cells was observed only in the late secretory phase, suggesting that accumulation of MIP-1alpha in epithelium occurred by self production and not via a receptor mediated pathway. MIP-1alpha was released from the denuded epithelium during menstruation and appeared to contribute to the accumulation of monocytes/macrophages into the endometrial cavity. MIP-1alpha has a number of biological effects other than monocytic chemotaxis, and some of these effects may be exerted in the endometrial tissue.
Matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes, capable of degrading proteins found in the extracellular matrix. MMPs 2 and 9 are known to be produced by microglia, the resident macrophages of the central nervous system. The control of the secretion of these proteases and the activation of proenzymes by other proteases such as plasmin, as well as the balance between MMP secretion and the secretion of their natural inhibitors (TIMPs), have an important relevance in the pathogenesis of multiple sclerosis (MS). The in vitro control of MMPs 2 and 9, TIMPs 1 and 2, and urokinase-type plasminogen activator by microglia was examined in response to a panel of chemokines (chemotactic cytokines), using ELISA and zymography techniques. The chemokines MCP1, MIP1beta, RANTES, IL-8, and Fractalkine were all found significantly to increase the secretion of MMPs and TIMPs by a human foetal microglial cell line, CHME3, after 24 h stimulation. The chemokines tested, MCP1, MIP1beta, and Fractalkine, were also shown to increase MMP9 secretion by primary isolated rat brain microglia in vitro. MCP1, MIP1alpha/beta, and RANTES significantly decreased the secretion of uPA into culture supernatants in ELISA experiments. These findings suggest an important potential role for the involvement of chemokines in the breakdown of the blood-brain barrier and also the destruction of myelin basic protein in MS.
Eosinophils are present in human endometrium only immediately before and during menstruation, suggesting a role in that process. The expression of the eosinophil chemoattractant, eotaxin, and its receptor, CCR3, within the human endometrium were investigated by immunohistochemical analysis of tissue sections spanning the entire menstrual cycle. Eotaxin was localized to perivascular cells in the late secretory phase, and it was also identified in eosinophils. However, the highest levels of this chemokine were present in both luminal and glandular epithelial cells during the proliferative and secretory phases of the cycle. Treatment of endometrial tissue with monensin, which blocks protein secretion, increased epithelial immunoreactive eotaxin, substantiating synthesis in these cells. Although the CCR3 receptor was expressed by eosinophils, it was also strongly expressed by endometrial epithelial cells. The CCR3 receptor on purified, cultured endometrial epithelial cells was functional, as assessed by a transient Ca(2+) flux in response to eotaxin. These analyses demonstrate that eotaxin is expressed by endometrial cells and may therefore be involved in the recruitment of eosinophils into this tissue premenstrually. However, the observation that this chemokine and the CCR3 molecule are strongly expressed by epithelial cells throughout the cycle suggests that these proteins may have additional important functions within the endometrium.
Recombinant fractalkine possesses both chemoattractive and adhesive properties in vitro. Previous studies have demonstrated an upregulation of this molecule on the membranes of activated human endothelial cells and hypothesised that fractalkine plays a role in the recruitment and adherence of monocytes to the activated endothelium. Here we present data analysing both the adhesive and chemoattractive properties of this chemokine expressed by activated human umbilical vein endothelial cells. We demonstrate that both recombinant fractalkine and endogenously produced fractalkine function as adhesion molecules, tethering monocytes to the endothelium. However, our data demonstrate that although recombinant fractalkine has the potential to function as a potent monocyte chemoattractant, the endogenous fractalkine cleaved from activated human umbilical vein endothelial cells is not responsible for the observed chemotaxis in this model. Instead, we show that monocyte chemoattractant protein-1 (MCP-1), secreted from the activated human umbilical vein endothelial cells, is responsible for the chemotaxis of these monocytes.
The endometrium contains many leukocytes, including macrophages, the numbers varying with the time of the menstrual cycle and being maximal peri-menstrually. The long-acting progestogenic contraceptive Norplant, has a high rate of discontinuation due to uterine bleeding; this is associated with large numbers of endometrial macrophages. Monocyte chemotactic proteins (MCP) act to recruit and activate monocytes into sites of inflammation. This study compared the cellular localization of endometrial MCP-1 and MCP-2 across the normal menstrual cycle and in users of Norplant. Both MCP-1 and MCP-2 were present in normal endometrium, but with very different patterns of cellular location and considerable variability between individuals. MCP-1 of epithelial origin was present in 77% of tissues, while stromal staining was present in 52% and vascular staining in 34% of samples. MCP-1 was also released from both epithelial and stromal cells in culture. MCP-2 staining was predominantly epithelial and was found in 52% of tissues while stromal staining was present in only 3/56 samples. Vascular staining of MCP-2 was found in 2/56 samples. The epithelial staining was mostly punctate and sometimes within uterine secretions. No correlation of staining for MCP-1 or -2 with the phase of the cycle was found in any cellular compartment. Very little immunoreactive MCP-1 or MCP-2 was detected in endometrium from Norplant users regardless of morphological subtype. These distributions do not support a role for either MCP-1 or MCP-2 in the migration of macrophages into the endometrium and suggest that these cytokines may have other functions in this tissue.
We know that the implantation process requires a functionally normal embryo at the blastocyst stage and a receptive endometrium, but also a communication link between them is needed. This paracrine dialogue between the embryo, endometrium and the corpus luteum are known to occur in ruminants and primates, more specifically endometrial-embryonic interactions have been reported in rodents and primates but not in humans. This process is a highly regulated mechanism and many molecules take part in this cross-talk. Here, we present updated information in humans on the embryonic regulation of endometrial epithelial molecules such as chemokines, adhesion and anti-adhesion molecules, and leptin during the apposition and adhesion phases of human implantation.
Chemokines are increasingly recognized as important regulators of uterine function.
The following is a review of uterine chemokines, especially monocyte chemotactic protein (MCP)-1, interleukin (IL)-8, and regulated-upon-activation normal-T-cell-expressed and -secreted (RANTES) protein, in reproductive physiology and pathology.
It is increasingly clear that IL-8, MCP-1, RANTES and their receptors are produced by endometrial, myometrial, and trophoblast cell types in a timed and co-ordinated manner. In addition to the regulation of leukocyte migration and function, uterine chemokines also display specific roles in endometrial angiogenesis, apoptosis, proliferation, and differentiation. IL-8, MCP-1 and RANTES are regulated by local growth factors and cytokines such as tumor necrosis factor-alpha (TNF-alpha), interferon-gamma, and IL-1. IL-8 takes part in cervical ripening and parturition. IL-8, MCP-1 and RANTES are also found at high levels in the peritoneal fluid of women with endometriosis.
Co-ordination of chemokine-chemokine receptor interactions plays an important role in the menstrual cycle and successful pregnancy. Moreover, unbalanced chemokine expression contributes to pathologic conditions typified by uncontrolled cellular proliferation, migration and invasion.
Recent studies indicate that chemoattractant cytokines (chemokines), together with tissue-specific adhesion molecules, coordinate the migration of antibody-secreting cells (ASCs) from their sites of antigen-driven differentiation in lymphoid tissues to target effector tissues. Developing ASCs downregulate the expression of receptors for lymphoid tissue chemokines and selectively upregulate the expression of chemokine receptors that might target the migration of IgA ASCs to mucosal surfaces, IgG ASCs to sites of tissue inflammation and both types of ASC to the bone marrow - an important site for serum antibody production. By directing plasma-cell homing, chemokines might help to determine the character and efficiency of mucosal, inflammatory and systemic antibody responses.
In human pregnancy, the embryo implants into the specialized mucosal wall of the uterus (decidua) and the placenta starts to form. Cells from the placenta (trophoblasts) invade into the uterine mucosa in order to open up maternal uterine arteries to ensure an adequate supply of blood to the developing fetus. The trophoblasts have a unique immunological phenotype compared to most cells especially with regard to their expression of major histocompatibility complex (MHC) antigens. On the other side of the interaction, the uterine mucosa (endometrium) differentiates in preparation for implantation. One of the changes that takes place is the appearance in the endometrium of a large number of maternal leukocytes in the final part of the menstrual cycle. If pregnancy ensues, these leukocytes continue to increase in number and are found in close contact with trophoblasts. The composition of this population of maternal immune cells is unusual compared to that seen at other mucosal sites. A lot of research has focused on whether maternal T-cell responses are suppressed or modified during pregnancy. Research has also concentrated on the specialized uterine natural killer (NK) cells, which are found in the decidua in large numbers during early pregnancy. These uterine NK cells have been shown to express receptors for trophoblast MHC antigens, but their role in pregnancy is still mysterious. The purpose of this review is to give an overview of what is known about the immunology at the implantation site and also to provide an update of some of the most recent findings in this field.
Recent studies suggest involvement of the immune system, including leukocytes and cytokines/chemokines, in various ovarian functions such as ovulation. Using the RT-PCR method, we examined expression of various chemokines and their receptors in normal mouse ovaries. Among seventeen examined chemokines (17 CC types and two CXC types), expressions of CC types MCP-1 and RANTES, and CXC type IP-10 were detected at high levels, while most CC types expressed at variable or low levels. Only five chemokines were not detected in the ovary. We next examined expression of chemokine receptors. CCR1 and CCR2, which are the receptors for MCP-1 and RANTES, were also expressed at constitutively high levels while others were not detectable. We further showed that a significant part of expression of both detected chemokines and receptors originated from peripheral blood leukocytes (PBL) circulating in the ovary. However, ovarian tissue was the major contributor of expression. Constitutive expression of several chemokines and their receptors suggests frequent migrations/movements of leukocytes in the ovary, which may be involved in ovarian functions other than ovulation.
Fetal cytotrophoblasts colonize the decidual spiral arteries during pregnancy and partially replace the endothelium by an as yet unknown mechanism. To clarify this issue, we cocultured trophoblast cells (TCs) and decidual endothelial cells (DECs) isolated from first trimester placentae and found by electron microscopic analysis that TCs adhered to DECs and migrated through the interendothelial junctions within 24 h. Since extravillous TCs were shown by FACS analysis to express vascular-endothelial (VE)-cadherin and platelet endothelial cell adhesion molecule-1 (PECAM)-1, we investigated the role of these junctional molecules in TC adhesion to DECs and transendothelial migration of cytotrophoblasts. Both VE-cadherin and PECAM-1 were present at the contact sites between TCs and DECs in decidual sections. TC adhesion and migration were markedly inhibited by mAbs to VE-cadherin and marginally by mAb to PECAM-1. Increased expression of VE-cadherin was observed at the contact areas between TCs and DECs, whereas PECAM-1 was found to be redistributed from intercellular junctions. The induction of apoptosis of DECs by TCs, as the mechanism responsible for their replacement, was ruled out by the negative staining with TUNEL of DECs cocultured with TCs and the absence of DNA fragmentation. In conclusion, VE-cadherin is involved in transendothelial migration of TCs, and replacement of DECs by TCs is not the result of apoptosis.
Leukocytes are critical mediators of endometrial remodeling, but the mechanisms by which leukocyte subpopulations enter the uterus are currently unknown. Endometrial leukocytes have no genomic progesterone receptors; thus, we hypothesized that leukocyte migration is induced indirectly by progesterone-regulated chemokines. Fractalkine (CX3CL1), a chemotactic membrane-bound adhesion factor, and its receptor (CX3CR1) were assessed by immunohistochemistry in endometrial samples across the menstrual cycle, in early pregnancy, and in women using progestin-only contraceptives. Fractalkine was localized predominantly to glandular epithelial and decidualized stromal cells, with the highest staining intensity in the secretory phase and early pregnancy. It was also detected in subpopulations of endometrial leukocytes (macrophages and uterine NK cells), with maximal numbers during the proliferative phase and early pregnancy. CX3CR1 was similarly colocalized to the glandular epithelium and decidualized stromal cells, with the highest expression in the secretory phase. CX3CR1-positive leukocytes (macrophages and neutrophils) were in greatest abundance during the menstrual phase. In the endometrium of women using progestin-only contraceptives, immunoreactive fractalkine was markedly reduced in the glandular epithelium, but was increased in decidualized stroma and infiltrating leukocytes. These findings support a number of roles for fractalkine in the endometrium, in the secretory phase, in early pregnancy, and when influenced by progestin-only contraceptives.
To determine whether primary human uterine epithelial cells in culture are able to influence monocyte chemotaxis and to establish whether the causal agent of chemotaxis is monocyte chemotactic protein (MCP)-1.
Tissue culture study.
University medical center.
Women aged 23 to 53 years who were undergoing hysterectomy (n=7).
Primary human endometrial epithelial cells were acquired from surgical specimens and grown to confluence and high transepithelial resistance. Conditioned media from epithelial cultures were analyzed for the presence of MCP-1 and for capacity to affect monocyte chemotaxis using the THP-1 monocyte line. Antibody neutralization of conditioned media was used to establish the role of MCP-1 in chemotaxis.
Assay of conditioned media for MCP-1, quantitative measurement of monocyte chemotaxis to conditioned media, and inhibition of chemotaxis by antibody neutralization of MCP-1.
Primary endometrial epithelial cells in monolayer culture secrete MCP-1 to both the apical and basolateral compartments. Monocyte chemotactic protein-1 was identified as the primary agent of monocyte chemotaxis by antibody neutralization.
These findings suggest that biologically active MCP-1 is secreted into both the uterine lumen as well as the underlying stroma and that it mediates the presence of monocytes, macrophages, and other immune cells in the uterine endometrium.
Breast cancer is an example of a solid tumour which is well treated in the early stages of disease by surgical excision, but once metastatic spread has occurred, medical therapies (chemotherapy and radiotherapy) are highly toxic, expensive and palliative. It is known that certain tumours exhibit specific patterns of metastasis, chemokines may provide a molecular answer to this mystery. Chemokines and their receptors play important roles in the various stages of tumour development and metastasis. Chemokines interact with their specific receptors as well as interacting with the glycosaminoglycan (GAG) component of proteoglycan. We discuss the basic metastatic process and the involvement of chemokines in breast cancer biology. Finally, we summarize potential therapeutic applications of chemokines and chemokine/glycosaminoglycan interactions including chemokine agonists, antagonists, anti-sense therapy, immunotherapy and soluble GAGs, as well as future perspectives in this field.
Enrichment of uterine natural killer (uNK) cells occurs during pregnancy in many species. However, functions of uNK cells and regulation of their uterine homing are not fully defined. In mice and women, uNK cells contribute to angiogenesis, a role reviewed here and now addressed in a mammal with an alternative placental type.
To address lymphocyte functions, RNA from murine or porcine endometrium and lymphocytes purified from endometrium were analyzed using real-time or reverse transcription PCR. To address homing potential, human blood CD56(+) lymphocytes were evaluated using both RNA and functional adhesion to endothelium presented under shear force in frozen sections of gestation day 7 C57Bl/6J implantation sites. Women were serially sampled over a menstrual cycle or a clinical preparatory cycle for embryo transfer.
Activation of murine uNK cells is associated with much greater increases in transcription for Eomes than for T-bet (Tbx21). Lymphocytes from normal porcine implantation sites transcribe vascular endothelial growth factor, placental growth factor, interferon-gamma and hypoxia-inducible factor (HIF)-1alpha. In fertile women, increases in L-selectin- and alpha4-integrin-mediated interactions between CD56(+) cells and endothelium occur at luteinizing hormone (LH) surge (cycling women) to oocyte pick up or embryo transfer, then return to pre-LH levels.
Uterine lymphocytes may universally promote pregnancy-associated endometrial angiogenesis. Recruitment of uNK precursor cells from blood appears to occur in a window promoted by rising plasma estrogen and LH and limited by rising progesterone.
The current study describes a statistically significant increase in macrophages (CD68-positive cells) in the decidua of preeclamptic patients. To elucidate the regulation of this monocyte infiltration, expression of monocyte chemoattractant protein-1 (MCP-1) was assessed in leukocyte-free first trimester decidual cells. Confluent decidual cells were primed for 7 days in either estradiol or estradiol plus medroxyprogesterone acetate to mimic the decidualizing steroidal milieu of the luteal phase and early pregnancy. The medium was exchanged for a serum-free defined medium containing corresponding steroids +/- tumor necrosis factor (TNF)-alpha or interleukin (IL)-1beta. After 24 hours, enzyme-linked immunosorbent assay measurements indicated that the addition of medroxyprogesterone acetate did not affect MCP-1 output, whereas 10 ng/ml of TNF-alpha or IL-1beta increased output by 83.5-fold +/- 20.6 and 103.1-fold +/- 14.7, respectively (mean +/- SEM, n = 8, P < 0.05). Concentration-response comparisons revealed that even 0.01 ng/ml of TNF-alpha or IL-1beta elevated MCP-1 output by more than 15-fold. Western blotting confirmed the enzyme-linked immunosorbent assay results, and quantitative reverse transcriptase-polymerase chain reaction confirmed corresponding effects on MCP-1 mRNA levels. The current study demonstrates that TNF-alpha and IL-1beta enhance MCP-1 in first trimester decidua. This finding suggests a mechanism by which recruitment of excess macrophages to the decidua impairs endovascular trophoblast invasion, the primary placental defect of preeclampsia.
Human embryo implantation is a complex process involving blastocyst attachment to the endometrial epithelium and subsequent trophoblast invasion of the decidua. Chemokines, critical regulators of leukocyte migration, are abundant in endometrial epithelial and decidual cells at this time. We hypothesized that endometrial chemokines stimulate trophoblast invasion. Chemokine receptors CX3CR1 and CCR1 were immunolocalized in human first-trimester implantation sites, specifically to endovascular extravillous trophoblasts, but not to the invading interstitial EVTs (iEVTs), with weak staining also on syncytium. CCR3 was localized to invading iEVTs and to microvilli on the syncytial surface. Expression of CX3CL1 (fractalkine), CCL7 (MCP-3), and their receptors (CX3CR1, CCR1, CCR2, CCR3, and CCR5) mRNA was examined in cellular components of the maternal-embryonic interface by RT-PCR. Both chemokines were abundant in entire endometrium and placenta, endometrial cells (primary cultures and HES, a human endometrial epithelial cell line) and trophoblast cell lines (JEG-3, ACIM-88, and ACIM-32). Chemokine receptor mRNA was expressed by placenta and trophoblast cell lines: CCR1 by all trophoblast cell types, whereas CCR2, CCR3, and CX3CR1 were more variable. CX3CR1, CCR1, CCR2, and CCR5 were also expressed by endometrial cells. Migration assays used the trophoblast cell line most closely resembling extravillous cytotrophoblast (AC1M-88). Trophoblast migration occurred in response to CX3CL1, CCL14, and CCL4, but not CCL7. Endometrial cell-conditioned media also stimulated trophoblast migration; this was attenuated by neutralizing antibodies to CX3CL1 and CCL4. Thus, chemokines are expressed by maternal and embryonic cells during implantation, whereas corresponding receptors are on trophoblast cells. Promotion of trophoblast migration by chemokines and endometrial cell conditioned medium indicates an important involvement of chemokines in maternal-fetal communication.
Remodelling of the human endometrium occurs during the normal menstrual cycle. This process involves the disintegration of the superficial or functionalis layer of the endometrium following the fall in progesterone resulting from the demise of the corpus luteum and the reconstruction of a new layer without scarring. The degradative properties of matrix metalloproteinases (MMP) and their presence in the endometrium during remodelling events suggests that they are effector molecules in this process. The features of menstruation parallel those of an inflammatory response and the abundance of leukocytes in the endometrium prior to the onset of menstruation indicates a role for these cells in the remodelling process. This review examines the relationship between leukocytes and the local production and activation of MMP within the endometrium.