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.

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. Pro-inflammatory 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 placenta, a unique organ that only develops during pregnancy, is essential for nutrient, oxygen, and waste exchange between offspring and mother. Yet, despite its importance, the placenta remains one of the least understood organs and knowledge of early placental formation is particularly limited. Abnormalities in placental development result in placental dysfunction or insufficiency whereby normal placental physiology is impaired. Placental dysfunction is a frequent source of pregnancy loss in livestock, inflicting serious economic impact to producers. Though the underlying causes of placental dysfunction are not well-characterized, initiation of disease is thought to occur during establishment of functional fetal and placental circulation. A comprehensive understanding of the mechanisms controlling placental growth and vascularization is necessary to improve reproductive success in livestock. We propose chemokine C-X-C motif ligand 12 (CXCL12) signaling through its receptor CXCR4 functions as a chief coordinator of vascularization through direct actions on fetal trophoblast and maternal endometrial and immune cells. To investigate CXCL12–CXCR4 signaling on uteroplacental vascular remodeling at the fetal–maternal interface, we utilized a CXCR4 antagonist (AMD3100). On day 12 post-breeding in sheep, osmotic pumps were surgically installed and delivered either AMD3100 or saline into the uterine lumen ipsilateral to the corpus luteum for 14 days. On day 35 of ovine pregnancy, fetal/placental and endometrial tissues were collected, snap-frozen in liquid nitrogen, and uterine horn cross sections were preserved for immunofluorescent analysis. Suppressing CXCL12–CXCR4 at the fetal–maternal interface during initial placental vascularization resulted in diminished abundance of select angiogenic factors in fetal and maternal placenta on day 35. Compared to control, less vascular endothelial growth factor (VEGF) and VEFG receptor 2 (KDR) were observed in endometrium when CXCL12–CXCR4 was diminished. Less VEGF was also evident in fetal placenta (cotyledons) in ewes receiving AMD3100 infusion compared to control. Suppressing CXCL12–CXCR4 at the fetal–maternal interface also resulted in greater autophagy induction in fetal and maternal placenta compared to control, suggestive of CXCL12–CXCR4 impacting cell survival. CXCL12–CXCR4 signaling may govern placental homeostasis by serving as a critical upstream mediator of vascularization and cell viability, thereby ensuring appropriate placental development.

Pregnancy detection in cattle can be expensive and labor intensive to producers. A simple, cost-efficient method of determining early pregnancy, chute side, is lacking within the industry. This study tested various proven pregnancy detection methods against an alternative method. Pregnancy was assessed by ultrasonography, blood serum pregnancy-specific protein B (PSPB), and urine estrone sulfate (“P-Test”). A fixed timed AI (FTAI) protocol was conducted on 14 predominantly British cattle housed at Tarleton State University in Stephenville, TX. Cows were synchronized using the co-synch plus 7 day CIDR protocol, with PGF injection of d 7. Heifers were synchronized similarly with the exception of GnRH injection on d 1. Estrotech patch scores (1 – 4, 1 showing little signs of estrus to 4 presenting signs of estrus) were utilized to indicate estrus activity prior to FTAI (21.4% vs. 78.6% females scored 1 vs. 4, respectively). FTAI was performed on protocol d 10 along with a 2mL injection of GnRH. Serum for PSPB analysis was collected at d 30 and 60 post insemination, while urine was sampled on d 60. Overall 53.8% (ultrasonography) and 71.4% (PSPB) of cattle were determined to be pregnant at d 30 post FTAI. Chi squared analysis proved that PSPB and ultrasound were similar at detecting pregnancy (P = 0.04). At d 60 “P-Test” color varied indicating differing levels of estrone sulfate. A t-test revealed increased PSPB from d 30–60 post FTAI (P < 0.0001); however, “P-Test” could not predict similar results (P = 0.655). Data suggest the “P-Test” is not a consistent method to diagnose early pregnancy. Future studies may be warranted to investigate a more desirable time point for utilizing the chute side “P-Test” as a reliable method to determine pregnancy.