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Early pregnancy features complex signaling between fetal trophoblast cells and maternal endometrium directing major peri-implantation events including localized inflammation and remodeling to establish proper placental development. Proinflammatory mediators are important for conceptus attachment, but a more precise understanding of molecular pathways regulating this process is needed to understand how the endometrium becomes receptive to implantation. Both chemokine ligand 12 (CXCL12) and its receptor CXCR4 are expressed by fetal and maternal tissues. We identified this pair as a critical driver of placental angiogenesis, but their additional importance to inflammation and trophoblast cell survival, proliferation, and invasion imply a role in syncytia formation at the fetal-maternal microenvironment. We hypothesized that CXCL12 encourages both endometrial inflammation and conceptus attachment during implantation. We employed separate ovine studies to (1) characterize endometrial inflammation during early gestation in the ewe, and (2) establish functional implications of CXCL12 at the fetal-maternal interface through targeted intrauterine infusion of the CXCR4 inhibitor AMD3100. Endometrial tissues were evaluated for inflammatory mediators, intracellular signaling events, endometrial modifications, and trophoblast syncytialization using western blotting and immunohistochemistry. Endometrial tissue from ewes receiving CXCR4 inhibitor demonstrated dysregulated inflammation and reduced AKT and NFKB, paired with elevated autophagic activity compared to control. Immunohistochemical observation revealed an impairment in endometrial surface remodeling and diminished trophoblast syncytialization following localized CXCR4 inhibition. These data suggest CXCL12-CXCR4 regulates endometrial inflammation and remodeling for embryonic implantation, and provide insight regarding mechanisms that, when dysregulated, lead to pregnancy pathologies such as intrauterine growth restriction and preeclampsia., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Problem: Chemokines help coordinate inflammation within the fetal-maternal microenvironment during gestation. The chemokine CXCL12 signaling through its receptor CXCR4 regulates inflammatory activity, but this phenomenon is not well understood during pregnancy, and there are no reports exploring the role of this pair in peripheral immune tolerance during gestation. Herein, we hypothesize that intrauterine CXCL12-CXCR4 signaling governs local and systemic immunomodulatory dynamics during early gestation in ewes., Method of Study: Osmotic pumps were surgically installed for intrauterine infusion of a CXCR4 inhibitor, AMD3100, beginning on day 12 post-breeding in sheep. Endometrial tissues were collected on day 35 of gestation and evaluated for inflammatory potential, Akt pathway activation, and autophagy induction. Demonstrative of peripheral immune activity, levels of select cytokines were assessed in daily blood samples collected throughout the study, as well as in corpus luteum and spleen on day 35., Results: Anti-inflammatory IL10 was primarily localized to endometrial glandular epithelium with lower abundance when CXCR4 was antagonized. Inhibition of CXCR4 at the fetal-maternal interface resulted in less activation of Akt in endometrium, while evidence of autophagy induction was greater. Corpora lutea from ewes receiving intrauterine AMD3100 exhibited lower interferon-gamma (IFNG) expression. Blood inflammatory potential was differentially altered in a temporal fashion throughout infusion. IL10 abundance in spleen was greater following CXCR4 inhibition at the fetal-maternal interface, while IFNG was less., Conclusion: Intrauterine CXCL12-CXCR4 signaling governs endometrial and systemic inflammation; disruption of this axis may have detrimental impacts on offspring and maternal health., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Placenta development is characterized by extensive angiogenesis and vascularization but if these processes are compromised placental dysfunction occurs, which is the underlying cause of pregnancy complications such as preeclampsia and intrauterine growth restriction. Dysregulation of placental angiogenesis has emerged as one of the main pathophysiological features in the development of placental insufficiency and its clinical consequences. The signaling axis initiated by chemokine ligand 12 (CXCL12) and its receptor CXCR4 stimulates angiogenesis in other tissues, and may be central to placental vascularization. We hypothesized that CXCL12-CXCR4 signaling governs the pro-angiogenic placental microenvironment by coordinating production of central angiogenic factors and receptors and regulates endometrial cell survival essential for placental function and subsequent fetal longevity. The CXCR4 antagonist, AMD3100, was used to elucidate the role of CXCL12-CXCR4 signaling regarding uteroplacental vascular remodeling at the fetal-maternal interface. On day 12 postbreeding, osmotic pumps were surgically installed and delivered either AMD3100 or PBS into the uterine lumen ipsilateral to the corpus luteum. On day 20, endometrial tissues were collected, snap-frozen in liquid nitrogen, and uterine horn cross sections preserved for immunofluorescent analysis. In endometrium from ewes receiving AMD3100 infusion, the abundance of select angiogenic factors was diminished, while presence of CD34+ cells increased compared to control ewes. Ewes receiving AMD3100 infusion also exhibited less activation of Akt/mTOR signaling, and elevated LC3B-II, a marker of cellular autophagy in endometrium. This study suggests that CXCL12-CXCR4 signaling governs placental homeostasis by serving as a critical upstream mediator of vascularization and cell viability, thereby ensuring appropriate placental development., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Placental dysfunction originates during placental development, specifically the events of placentation. Vascularization is a key component of placentation due to its role in fetal-maternal exchange. Cytokines are regulators of placental development; signaling of chemokine ligand 12 (CXCL12) and its receptor (CXCR4) drives placental vascularization. Utilizing an in vivo sheep model, we demonstrated that suppressing CXCL12-CXCR4 signaling at the fetal-maternal interface reduces placental vascularization. We hypothesized these negative impacts early in gestation would result in compromised placental development and vascularization at mid to late gestation. On day 12 post-breeding, osmotic pumps were surgically installed in 37 ewes and delivered either CXCR4 inhibitor (AMD3100) at 1x dose (n = 8),1.5X dose (n = 8), or 3X dose (n = 8) or saline (n = 13) into the uterine lumen ipsilateral to the corpus luteum for 14 days. On day 90 and day 135 placental tissues were collected and maternal (caruncle) and fetal (cotyledon) placenta components were separated and analyzed. Gene expression of angiogenic factors were analyzed using Real-time qPCR. Gene expression was significantly altered by AMD3100 treatment for the vascular endothelial growth factor receptors (FLT-1 and KDR) primarily in day 90 placenta contralateral to the corpus luteum. Significant decreases in KDR were seen in 3x compared to control (P < 0.03), 3x compared to 1.5x (P < 0.008), and 3x contralateral compared to ipsilateral (P < 0.01); however, FLT-1 significantly increased (P < 0.02) from control to 1.5x. Day 90 caruncle tissue had significant increases in FLT-1 from control to 3x (0.02), 1.5x to 3x (P < 0.03); however, 1.5x treatment FLT-1 significantly decreased (P < 0.02) ipsilateral to contralateral. Fibroblast growth factor 2 increased in both contralateral 3x compared to control cotyledon (P < 0.03) and caruncle (P < 0.03). Our data underscore the importance of CXCL12-CXCR4 signaling during placentation and provide strong evidence that altering CXCR4-mediated signaling during early placentation induces enduring effects altering vascular development later in gestation. A greater understanding of CXCL12/CXCR4 functions may reveal methods to improve reproductive success and fetal health. [ABSTRACT FROM AUTHOR]

A proper balance of inflammatory cytokines at the fetal-maternal interface is crucial for successful embryo implantation and placental formation. Chemokine ligand 12 (CXCL12) and receptor CXCR4 are expressed by fetal and maternal placental tissues and implicated in cytokine synthesis. However, mechanisms controlling the axis and placental inflammatory profile are not well characterized. Using an in-vivo sheep model, we previously demonstrated suppressing CXCL12-CXCR4 signaling at the fetal-maternal interface during implantation yielded dysregulated production of inflammatory cytokines in the placenta. Our objective was to determine if disrupting the cytokine profile during implantation alters the placental inflammatory profile at mid-to-late gestation. Day 12 post-breeding, osmotic pumps were surgically installed in 37 ewes to deliver CXCR4 inhibitor (1.5X dose, n = 8; 1X dose, n = 8; 3X dose, n = 8) or saline (n = 13) into the uterine lumen ipsilateral to the corpus luteum for 14 days. On days 90 and 135 of gestation, placental tissues were collected; maternal (caruncle) and fetal (cotyledon) placenta components were then separated and analyzed. Real-time qPCR was used to measure gene expression of pro-inflammatory cytokines tumor necrosis factor (TNF), interferon-γ (IFNγ), interleukin-12 (IL12), and anti-inflammatory cytokines interleukin-10 (IL10) and transforming growth factor-β (TGFβ). Gene expression for TGFβ from tissues ipsilateral to the pump on day 90 increased (P < 0.03) in caruncles from 3x treated ewes compared to control ewes while IL10 mRNA decreased (P < 0.02) in cotyledons from 1.5x compared to 3x treatment. Furthermore, analysis of day 135 1x treated ewes showed greater expression (P < 0.03) of IL12 in caruncles ipsilateral compared to contralateral of the pump; meanwhile IL10 showed decreased expression (P < 0.02) in cotyledons ipsilateral compared to contralateral of the pump. High levels of TGFβ, IFNγ (whose production is promoted by increased IL12), and TNF along with decreased levels of IL10 have been associated with preeclampsia in women. The changes in gene expressions of ovine immune cytokines with a disrupted CXCL12-CCR4 axis may be related to preeclamptic events. Ultimately, modulating CXCL12- induced actions is a novel approach to manipulating the fetal-maternal environment when most pregnancy losses occur and may reveal methods to improve reproductive success and fetal health in livestock. [ABSTRACT FROM AUTHOR]

A proper balance of pro and anti-inflammatory cytokines at the fetal-maternal interface is crucial for successful embryo implantation and placental formation (placentation) during early gestation. However, mechanisms controlling this inflammatory profile are not well characterized. The C-X-C motif chemokine ligand 12 (CXCL12) and its receptor CXCR4 are expressed by fetal and maternal placental tissues and implicated in cytokine synthesis; roles of this axis during placentation are unclear. Using an in vivo sheep model, we demonstrated that suppressing CXCL12-CXCR4 signaling at the fetal-maternal interface during implantation results in dysregulated production of inflammatory cytokines in the placenta. The objective of the current study was to determine if the disrupted cytokine profile from suppressing CXCL12-CXCR4 signaling early in gestation would result in altered placental inflammatory profile at midgestation. On day 12 post-breeding, osmotic pumps were surgically installed in 37 ewes and delivered either CXCR4 inhibitor (AMD3100) at 1X dose, (n=8), 1.5X, (n=8), or 3X, (n=8) or saline (n=13) into the uterine lumen ipsilateral to the corpus luteum for 14 days. On days 90 and 135, placental tissues were collected, and maternal and fetal placenta components separated and analyzed. Gene expression for select cytokines were analyzed in the placenta using Real-time qPCR. On day 90, mRNA expression was significantly altered by AMD3100 treatment for tumor necrosis factor (TNF), interleukin 12 (IL-12), and transforming growth factor beta (TGFB) in fetal placenta. Expression of TGFB in fetal placenta was also altered by AMD3100 treatment compared to control on day 135 whereas IL-12 expression was significantly altered in maternal placenta on day 135. In conclusion, modulating CXCL12-induced actions is a novel approach to manipulating the fetalmaternal environment when most pregnancy losses occur. Increasing our understanding of CXCL12-induced actions controlling the placental inflammatory environment will hopefully reveal methods to improve reproductive success and fetal health in livestock. [ABSTRACT FROM AUTHOR]