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Two experiments evaluated the effects of an injectable trace mineral (ITM) solution at weaning on trace mineral (TM) status, inflammatory and antioxidant responses, grazing behavior, response to vaccination, and growth of beef calves. Experiment 1 used 86 Nellore calves (40 females and 46 males; body weight [BW] = 198 ± 30.8 kg; 8 ± 1 mo of age) weaned (day 0) and assigned into one of two treatments: saline (0.9% NaCl) or ITM (60 mg of Zn/mL, 15 mg of Cu/mL, 5 mg of Se/mL, and 10 mg of Mn/mL). Saline and ITM were administered subcutaneously at a dose of 1 mL/45 kg of BW. On day 0, calves were vaccinated against infectious bovine rhinotracheitis (IBR), parainfluenza-3 (PI 3 ), bovine viral diarrhea virus types 1 and 2 (BVDV-1 and 2) and bovine respiratory syncytial virus (BRSV). Blood samples and BW were collected on days 0, 3, 8, 15, 51, and 100, and grazing behavior was evaluated on days 1, 2, 4, 5, 6, 7, and 9. The ITM did not affect ( P  ≥ 0.56) serum mineral concentrations of Zn and Cu, but decreased ( P  ≤ 0.02) plasma concentrations of cortisol on days 3 and 8 and haptoglobin on day 3. The ITM increased ( P  < 0.01) plasma concentration of superoxide dismutase on days 8, 15, and 51 and tended to decrease ( P  = 0.08) plasma concentration of glutathione peroxidase on day 3. Furthermore, there were no effects of treatment ( P  ≥ 0.14) on most of the behavior variables evaluated, ITM reduced ( P  = 0.01) the seeking time on days 0 and 4. Furthermore, ITM tended to increase ( P  = 0.10) the serum titer concentration against IBR on days 15 and 51 but did not affect ( P  ≥ 0.12) titer concentration against PI 3 , BVDV-1, and 2, and growth. Experiment 2 used 50 Brangus male calves (BW = 264 ± 34.1 kg; 8 ± 1 mo of age) weaned on day 0, vaccinated against respiratory diseases, stratified by BW, and randomly assigned to saline or ITM as described in experiment 1. Liver samples were collected on days 0, 14, and 197, blood samples on days 0, 14, and BW on days 0, 14, 44, 78, 122, 162, and 197. The ITM increased ( P  ≤ 0.03) the liver concentration of Cu and Se on day 14 but did not affect ( P  ≥ 0.17) the liver concentration of Zn and Mn. In addition, ITM increased ( P  = 0.05) the serum titer concentration against BVDV-2 but did not affect ( P  ≥ 0.20) the titer concentrations against IBR, PI 3 , BVDV-1, and BRSV, and did not affect ( P  ≥ 0.29) the growth. In conclusion, ITM application at weaning improved Cu and Se status, enhanced antioxidant and immune responses, and reduced stress and inflammation in calves, though it did not affect growth., Competing Interests: The authors declare that they have no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Maternal malnutrition gives rise to both short- and long-term consequences for the survival and health of the offspring. As the intermediary between mother and fetus, the placenta has the potential to interpret environmental signals, such as nutrient availability, and adapt to support fetal growth and development. While this potential is present, it is clear that at times placental adaptation fails to occur resulting in poor pregnancy outcomes. This review will focus on placental responses to maternal undernutrition related to changes in placental vascularization and hemodynamics and placental nutrient transport systems across species. While much of the available literature describes placental responses that result in poor fetal outcomes, novel models have been developed to utilize the inherent variation in fetal weight when dams are nutrient restricted to identify placental adaptations that result in normal-weight offspring. Detailed analyses of the spectrum of placental responses to maternal malnutrition point to alternations in placental histoarchitectural and vascular development, amino acid and lipid transport mechanisms, and modulation of immune-related factors. Dietary supplementation with selected nutrients, such as arginine, has the potential to improve placental growth and function through a variety of mechanisms including stimulating cell proliferation, protein synthesis, angiogenesis, vasodilation, and gene regulation. Improved understanding of placental responses to environmental cues is necessary to develop diagnostic and intervention strategies to improve pregnancy outcomes.

Venous malformations (VMs) are slow-flow malformations of the venous vasculature and are the most common type of vascular malformation with a prevalence of 1%. Germline and somatic mutations have been shown to contribute to VM pathogenesis, but how these mutations affect VM pathobiology is not well understood. The goal of this study was to characterize VM endothelial and mural cell expression by performing a comprehensive expression analysis of VM vasculature. VM specimens (n = 16) were stained for pan-endothelial, arterial, venous, and endothelial progenitor cell proteins; proliferation was assessed with KI67. Endothelial cells in the VM vessels were abnormally orientated and improperly specified, as seen by the misexpression of both arterial and endothelial cell progenitor proteins not observed in control vessels. Consistent with arterialization of the endothelial cells, VM vessels were often surrounded by multiple layers of disorganized mural cells. VM endothelium also had a significant increase in proliferative endothelial cells, which may contribute to the dilated channels seen in VMs. Together the expression analysis indicates that the VM endothelium is misspecified and hyperproliferative, suggesting that VMs are biologically active lesions, consistent with clinical observations of VM progression over time., Competing Interests: The authors have declared that no competing interests exist.

Methylation of histone H3K9 is a hallmark of epigenetic silencing in eukaryotes. Nucleosome modifications often rely on positive feedback where enzymes are recruited by modified nucleosomes. A combination of local and global feedbacks has been proposed to account for some dynamic properties of heterochromatin, but the range at which the global feedbacks operate and the exact mode of heterochromatin propagation are not known. We investigated these questions in fission yeast. Guided by mathematical modeling, we incrementally increased the size of the mating-type region and profiled heterochromatin establishment over time. We observed exponential decays in the proportion of cells with active reporters, with rates that decreased with domain size. Establishment periods varied from a few generations in wild type to >200 generations in the longest region examined, and highly correlated silencing of two reporters located outside the nucleation center was observed. On a chromatin level, this indicates that individual regions are silenced in sudden bursts. Mathematical modeling accounts for these bursts if heterochromatic nucleosomes facilitate a deacetylation or methylation reaction at long range, in a distance-independent manner. A likely effector of three-dimensional interactions is the evolutionarily conserved Swi6 HP1 H3K9me reader, indicating the bursting behavior might be a general mode of heterochromatin propagation., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Obesity is associated with altered glycine metabolism in humans. This study investigated the mechanisms regulating glycine metabolism in obese rats. Eight-week-old Zucker diabetic fatty rats (ZDF; a type-II diabetic animal model) received either 1% glycine or 1.19% L-alanine (isonitrogenous control) in drinking water for 6 weeks. An additional group of lean Zucker rats also received 1.19% L-alanine as a lean control. Glycine concentrations in serum and liver were markedly lower in obese versus lean rats. Enteral glycine supplementation restored both serum and hepatic glycine levels, while reducing mesenteric and internal white fat mass compared with alanine-treated ZDF rats. Blood glucose and non-esterified fatty acid (NEFA) concentrations did not differ between the control and glycine-supplemented ZDF rats (P > 0.10). Both mRNA and protein expression of aminomethyltransferase (AMT) and glycine dehydrogenase, decarboxylating (GLDC) were increased in the livers of obese versus lean rats (P < 0.05). In contrast, glycine cleavage system H (GCSH) hepatic mRNA expression was downregulated in obese versus lean rats, although there was no change in protein expression. These findings indicate that reduced quantities of glycine observed in obese subjects likely results from an upregulation of the hepatic glycine cleavage system and that dietary glycine supplementation potentially reduces obesity in ZDF rats.

Maternal nutrient restriction impairs placental growth and development, but available evidence suggests that adaptive mechanisms exist, in a subset of nutrient restricted (NR) ewes, that support normal fetal growth and do not result in intrauterine growth restriction (IUGR). This study utilized Affymetrix GeneChip Bovine and Ovine Genome 1.0 ST Arrays to identify novel placental genes associated with differential fetal growth rates within NR ewes. Singleton pregnancies were generated by embryo transfer and, beginning on Day 35 of pregnancy, ewes received either a 100% National Research Council (NRC) (control-fed group; n = 7) or 50% NRC (NR group; n = 24) diet until necropsy on Day 125. Fetuses from NR ewes were separated into NR non-IUGR ( n = 6) and NR IUGR ( n = 6) groups based on Day 125 fetal weight for microarray analysis. Of the 103 differentially expressed genes identified, 15 were upregulated and 88 were downregulated in NR non-IUGR compared to IUGR placentomes. Bioinformatics analysis revealed that upregulated gene clusters in NR non-IUGR placentomes associated with cell membranes, receptors, and signaling. Downregulated gene clusters associated with immune response, nutrient transport, and metabolism. Results illustrate that placentomal gene expression in late gestation is indicative of an altered placental immune response, which is associated with enhanced fetal growth, in a subpopulation of NR ewes.

Introduction: Maternal nutrient partitioning, uteroplacental blood flow, transporter activity, and fetoplacental metabolism mediate nutrient delivery to the fetus. Inadequate availability or delivery of nutrients results in intrauterine growth restriction (IUGR), a leading cause of neonatal morbidity and mortality. Maternal nutrient restriction can result in IUGR, but only in an unforeseeable subset of individuals., Methods: To elucidate potential mechanisms regulating fetal nutrient availability, singleton sheep pregnancies were generated by embryo transfer. Pregnant ewes received either a 50% NRC (NR; n = 24) or 100% NRC (n = 7) diet from gestational Day 35 until necropsy on Day 125. Maternal weight did not correlate with fetal weight; therefore, the six heaviest (NR Non-IUGR) and five lightest (NR IUGR) fetuses from nutrient-restricted ewes, and seven 100% NRC fetuses, were compared to investigate differences in nutrient availability., Results: Insulin, multiple amino acids, and their metabolites, were reduced in fetal circulation of NR IUGR compared to NR Non-IUGR and 100% NRC pregnancies. In contrast, glucose in fetal fluids was not different between groups. There was a nearly two-fold reduction in placentome volume and fetal/maternal interface length in NR IUGR compared to NR Non-IUGR and 100% NRC pregnancies. Changes in amino acid concentrations were associated with altered expression of cationic (SLC7A2, SLC7A6, and SLC7A7) and large neutral (SLC38A2) amino acid transporters in placentomes., Discussion: Results establish a novel approach to study placental adaptation to maternal undernutrition in sheep and support the hypothesis that amino acids and polyamines are critical mediators of placental and fetal growth in sheep., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: In recent decades, there has been a growing interest in the impact of insults during pregnancy on postnatal health and disease. It is known that changes in placental development can impact fetal growth and subsequent susceptibility to adult onset diseases; however, a method to collect sufficient placental tissues for both histological and gene expression analyses during gestation without compromising the pregnancy has not been described. The ewe is an established biomedical model for the study of fetal development. Due to its cotyledonary placental type, the sheep has potential for surgical removal of materno-fetal exchange tissues, i.e., placentomes. A novel surgical procedure was developed in well-fed control ewes to excise a single placentome at mid-gestation., Results: A follow-up study was performed in a cohort of nutrient-restricted ewes to investigate rapid placental changes in response to undernutrition. The surgery averaged 19 min, and there were no viability differences between control and sham ewes. Nutrient restricted fetuses were smaller than controls (4.7 ± 0.1 kg vs. 5.6 ± 0.2 kg; P  < 0.05), with greater dam weight loss (- 32.4 ± 1.3 kg vs. 14.2 ± 2.2 kg; P  < 0.01), and smaller placentomes at necropsy (5.7 ± 0.3 g vs. 7.2 ± 0.9 g; P  < 0.05). Weight of sampled placentomes and placentome numbers did not differ., Conclusions: With this technique, gestational studies in the sheep model will provide insight into the onset and complexity of changes in gene expression in placentomes resulting from undernutrition (as described in our study), overnutrition, alcohol or substance abuse, and environmental or disease factors of relevance and concern regarding the reproductive health and developmental origins of health and disease in humans and in animals., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

Infantile hemangioma (IH) is a vascular tumor that begins with rapid vascular proliferation shortly after birth, followed by vascular involution in early childhood. We have found that NOTCH3, a critical regulator of mural cell differentiation and maturation, is expressed in hemangioma stem cells (HemSCs), suggesting that NOTCH3 may function in HemSC-to-mural cell differentiation and pathological vessel stabilization. Here, we demonstrate that NOTCH3 is expressed in NG2+PDGFRβ+ perivascular HemSCs and CD31+GLUT1+ hemangioma endothelial cells (HemECs) in proliferating IHs and becomes mostly restricted to the αSMA+NG2loPDGFRβlo mural cells in involuting IHs. NOTCH3 knockdown in HemSCs inhibited in vitro mural cell differentiation and perturbed αSMA expression. In a mouse model of IH, NOTCH3 knockdown or systemic expression of the NOTCH3 inhibitor, NOTCH3 Decoy, significantly decreased IH blood flow, vessel caliber, and αSMA+ perivascular cell coverage. Thus, NOTCH3 is necessary for HemSC-to-mural cell differentiation, and adequate perivascular cell coverage of IH vessels is required for IH vessel stability.

Study Question: Do naturally occurring, hyperandrogenic (≥1 SD of population mean testosterone, T) female rhesus monkeys exhibit traits typical of women with polycystic ovary syndrome (PCOS)?, Summary Answer: Hyperandrogenic female monkeys exhibited significantly increased serum levels of androstenedione (A4), 17-hydroxyprogesterone (17-OHP), estradiol (E2), LH, antimullerian hormone (AMH), cortisol, 11-deoxycortisol and corticosterone, as well as increased uterine endometrial thickness and evidence of reduced fertility, all traits associated with PCOS., What Is Known Already: Progress in treating women with PCOS is limited by incomplete knowledge of its pathogenesis and the absence of naturally occurring PCOS in animal models. A female macaque monkey, however, with naturally occurring hyperandrogenism, anovulation and polyfollicular ovaries, accompanied by insulin resistance, increased adiposity and endometrial hyperplasia, suggests naturally occurring origins for PCOS in nonhuman primates., Study Design, Size, Duration: As part of a larger study, circulating serum concentrations of selected pituitary, ovarian and adrenal hormones, together with fasted insulin and glucose levels, were determined in a single, morning blood sample obtained from 120 apparently healthy, ovary-intact, adult female rhesus monkeys (Macaca mulatta) while not pregnant or nursing. The monkeys were then sedated for somatometric and ultrasonographic measurements., Participants/materials, Setting, Methods: Female monkeys were of prime reproductive age (7.2 ± 0.1 years, mean ± SEM) and represented a typical spectrum of adult body weight (7.4 ± 0.2 kg; maximum 12.5, minimum 4.6 kg). Females were defined as having normal (n = 99) or high T levels (n = 21; ≥1 SD above the overall mean, 0.31 ng/ml). Electronic health records provided menstrual and fecundity histories. Steroid hormones were determined by tandem LC-MS-MS; AMH was measured by enzymeimmunoassay; LH, FSH and insulin were determined by radioimmunoassay; and glucose was read by glucose meter. Most analyses were limited to 80 females (60 normal T, 20 high T) in the follicular phase of a menstrual cycle or anovulatory period (serum progesterone <1 ng/ml)., Main Results and the Role of Chance: Of 80 monkeys, 15% (n = 12) exhibited classifiable PCOS-like phenotypes. High T females demonstrated elevations in serum levels of LH (P < 0.036), AMH (P < 0.021), A4 (P < 0.0001), 17-OHP (P < 0.008), E2 (P < 0.023), glucocorticoids (P < 0.02-0.0001), the serum T/E2 ratio (P < 0.03) and uterine endometrial thickness (P < 0.014) compared to normal T females. Within the high T group alone, anogenital distance, a biomarker for fetal T exposure, positively correlated (P < 0.015) with serum A4 levels, while clitoral volume, a biomarker for prior T exposure, positively correlated (P < 0.002) with postnatal age. Only high T females demonstrated positive correlations between serum LH, and both T and A4. Five of six (83%) high T females with serum T ≥2 SD above T mean (0.41 ng/ml) did not produce live offspring., Large Scale Data: N/A., Limitations, Reasons for Caution: This is an initial study of a single laboratory population in a single nonhuman primate species. While two biomarkers suggest lifelong hyperandrogenism, phenotypic expression during gestation, prepuberty, adolescence, mid-to-late reproductive years and postmenopause has yet to be determined., Wider Implications of the Findings: Characterizing adult female monkeys with naturally occurring hyperandrogenism has identified individuals with high LH and AMH combined with infertility, suggesting developmental linkage among traits with endemic origins beyond humans. PCOS may thus be an ancient phenotype, as previously proposed, with a definable pathogenic mechanism(s)., Study Funding/competing Interest(s): Funded by competitive supplement to P51 OD011106 (PI: Mallick), by P50 HD028934 (PI: Marshall) and by P50 HD044405 (PI: Dunaif). The authors have no potential conflicts of interest., (© The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Background: Angiogenesis is an essential process in endometriosis disease progression. Earlier, we demonstrated that anti-angiogenic peptide, ABT-898 prevents neoangiogenesis of human endometriotic lesions in a xenograft mouse model. Since angiogenesis is essential for normal ovarian and uterine function, we evaluated effects of ABT-898 on normal female reproductive processes in mice., Methods: Cycling female C57BL/6N mice were dosed with ABT-898 (100 mg/kg) or 5 % dextrose control for 21 consecutive days to cover multiple estrous cycles (average estrous cycle 4 to 5 days in mice). Pregnant female mice were dosed with ABT-898 (100 mg/kg) or control on alternate days over the course of gestation, beginning at gestation day 7.5 to 17.5 (gestation length 21 days). Histological analysis along with CD31 and Vimentin immunohistochemistry were performed on ovaries and uteri obtained from treated and control mice. To understand the influence of ABT-898 on systemic angiogenic factors, a Pro Mouse Cytokine 9-plex assay was performed on plasma samples obtained from mice prior to treatment, during the second week of ABAT-898 or control treatment and on the last day of treatment., Results: ABT-898 did not affect the number of estrous cycles over the 21 day treatment compared to control. Histological analysis of ovaries found no difference in the number of primordial, primary, secondary, and antral follicles between ABT-898 treated and control groups. Similarly, no difference was observed in the microvessel density between ABT-898 treated and control uteri, ovarian follicles or corpus luteum when assessed using CD31 or vimentin immunohistochemistry. Electron microscopy revealed similar capillary structure and appearance in both ABT-898 treated and control uteri. Although peripheral blood angiogenic cytokine profiles (IL-15, IL-18, M-CSF, b-FGF, PDGF-bb, MIG, MIP-2, LIF and VEGF) changed over the course of the intervention, there was no significant difference between ABT-898 and control groups at any of the studied time points. Treatment with ABT-898 during pregnancy had no effect on litter size at birth, pup weight at birth or pup weight at weaning., Conclusion: Our findings suggest that ABT-898 may not alter angiogenesis dependent reproductive processes in female mice. However, an extensive reproductive toxicology screening is required to substantiate use of ABT-898 in future.

Objective: To determine the impact of endometriotic lesion removal on local and systemic inflammation., Design: Multiplex cytokine analysis on samples from endometriosis patients before surgery, 2 weeks after surgery, and 3 months after surgery., Setting: Academic teaching hospital and university., Patient(s): A total of 43 endometriosis patients before and after excision of lesions by means of laparoscopic surgery, and 25 normal women., Intervention(s): None., Main Outcome Measure(s): Plasma, eutopic and ectopic tissue, and peritoneal fluid cytokine levels., Result(s): Compared with presurgery plasma samples, levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL) 2, IL-8, and IL-10 decreased significantly by 2 weeks after surgery in endometriosis patients. Interestingly, levels began to rise at 3 months after surgery in most cases. In tissue, levels of GM-CSF and IL-15 were lower in eutopic tissue, while levels of basic fibroblast growth factor, interferon-inducible protein 10, IL-1 receptor antagonist, granulocyte colony-stimulating factor, macrophage inflammatory protein 1β, IL-7, and IL-5 were higher in eutopic than in ectopic tissue. In peritoneal fluid, levels of IL-5 and IL-12 were higher in early versus advanced stages of endometriosis. Compared with normal women, plasma from endometriosis patients had higher levels of inflammatory cytokines., Conclusion(s): Endometriotic lesion removal significantly alters the inflammatory profile both locally and systemically in women with endometriosis. Our findings indicate that ectopic lesions are the major drivers of systemic inflammation in endometriosis. The transitory nature of the change may reflect the recurrence of the condition and the influence of systemic factors in its onset., (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Unlabelled: Infantile hemangiomas (IHs) are the most common vascular tumor and arise from a hemangioma stem cell (HemSC). Propranolol has proved efficacious for problematic IHs. Propranolol is a nonselective β-adrenergic receptor (βAR) antagonist that can lower cAMP levels and activate the mitogen-activated protein kinase (MAPK) pathway downstream of βARs. We found that HemSCs express β1AR and β2AR in proliferating IHs and determined the role of these βARs and the downstream pathways in mediating propranolol's effects. In isolated HemSCs, propranolol suppressed cAMP levels and activated extracellular signal-regulated kinase (ERK)1/2 in a dose-dependent fashion. Propranolol, used at doses of <10(-4) M, reduced cAMP levels and decreased HemSC proliferation and viability. Propranolol at ≥10(-5) M reduced cAMP levels and activated ERK1/2, and this correlated with HemSC apoptosis and cytotoxicity at ≥10(-4) M. Stimulation with a βAR agonist, isoprenaline, promoted HemSC proliferation and rescued the antiproliferative effects of propranolol, suggesting that propranolol inhibits βAR signaling in HemSCs. Treatment with a cAMP analog or a MAPK inhibitor partially rescued the HemSC cell viability suppressed by propranolol. A selective β2AR antagonist mirrored propranolol's effects on HemSCs in a dose-dependent fashion, and a selective β1AR antagonist had no effect, supporting a role for β2AR signaling in IH pathobiology. In a mouse model of IH, propranolol reduced the vessel caliber and blood flow assessed by ultrasound Doppler and increased activation of ERK1/2 in IH cells. We have thus demonstrated that propranolol acts on HemSCs in IH to suppress proliferation and promote apoptosis in a dose-dependent fashion via β2AR perturbation, resulting in reduced cAMP and MAPK activation., Significance: The present study investigated the action of propranolol in infantile hemangiomas (IHs). IHs are the most common vascular tumor in children and have been proposed to arise from a hemangioma stem cell (HemSC). Propranolol, a nonselective β-adrenergic receptor (βAR) antagonist, has proven efficacy; however, understanding of its mechanism of action on HemSCs is limited. The presented data demonstrate that propranolol, via βAR perturbation, dose dependently suppresses cAMP levels and activated extracellular signal-regulated kinase 1/2. Furthermore, propranolol acts via perturbation of β2AR, and not β1AR, although both receptors are expressed in HemSCs. These results provide important insight into propranolol's action in IHs and can be used to guide the development of more targeted therapy., (©AlphaMed Press.)

Endometriosis is a chronic, inflammatory disease characterized by the growth of endometrial tissue in aberrant locations outside the uterus. Neoangiogenesis or establishment of new blood supply is one of the fundamental requirements of endometriotic lesion survival in the peritoneal cavity. IL-17A is emerging as a potent angiogenic and proinflammatory cytokine involved in the pathophysiology of several chronic inflammatory diseases such as rheumatoid arthritis and psoriasis. However, sparse information is available in the context of endometriosis. In this study, we demonstrate the potential importance of IL-17A in the pathogenesis and pathophysiology of endometriosis. The data show a differential expression of IL-17A in human ectopic endometriotic lesions and matched eutopic endometrium from women with endometriosis. Importantly, surgical removal of lesions resulted in significantly reduced plasma IL-17A concentrations. Immunohistochemistry revealed localization of IL-17A primarily in the stroma of matched ectopic and eutopic tissue samples. In vitro stimulation of endometrial epithelial carcinoma cells, Ishikawa cells, and HUVECs with IL-17A revealed significant increase in angiogenic (vascular endothelial growth factor and IL-8), proinflammatory (IL-6 and IL-1β), and chemotactic cytokines (G-CSF, CXCL12, CXCL1, and CX3CL1). Furthermore, IL-17A promoted tubulogenesis of HUVECs plated on Matrigel in a dose-dependent manner. Thus, we provide the first evidence, to our knowledge, that endometriotic lesions produce IL-17A and that the removal of the lesion via laparoscopic surgery leads to the significant reduction in the systemic levels of IL-17A. Taken together, our data show a likely important role of IL-17A in promoting angiogenesis and proinflammatory environment in the peritoneal cavity for the establishment and maintenance of endometriosis lesions., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Endometriosis is a gynecological disease defined by the growth of endometrium outside of the uterus. Although endometriosis contributes to 50% of female infertility cases, medical treatments are incompatible with pregnancy. Angiogenesis, the growth of blood vessels from existing vasculature, plays a crucial role in endometriotic lesion growth and survival. Previously, we demonstrated the effectiveness of thrombospondin-1 analog, ABT-898 (Abbott Laboratories) to inhibit endometriotic lesion vascularization in mice. We have now evaluated the trans-generational implications of ABT-898 treatment before and during mouse pregnancy. We hypothesized that ABT-898 would target lesion vasculature without affecting pregnancy, offspring development, or ovarian and uterine vascularity in mice. Endometriosis was induced using human endometrium in β-estradiol-primed BALB/c-Rag-2-/-Il2rγ-/- mice receiving intraperitoneal injections of ABT-898 (25 mg/kg) or 5% dextrose control for 21 days. Ultrasound assessment of lesion vascularization revealed a reduction in blood flow supplying treated lesions. Excised ABT-898 treated lesions stained for CD31+ endothelial cells exhibited a decrease in microvessel density. Following confirmation of estrous cycling, mice were bred and treated with ABT-898 on gestation days 7, 9, 11, 13, 15, 17, and 19. ABT-898 did not affect estrous cycling or pregnancy parameters including litter size across generations and offspring weight gain. Quantification of angiogenic cytokine plasma levels revealed no significant differences between treatment groups. Vimentin staining of the uterus and ovary revealed no observable effects of ABT-898. Similarly, no obvious histological anomalies were observed in the kidney, liver, ovary, or uterus following ABT-898 treatment. These results suggest that ABT-898 effectively inhibit endometriotic lesion vascularization without affecting trans-generational pregnancy outcomes in mice.

Endometrial lymphocytes are recruited to the porcine maternal-fetal interface by conceptus-derived signals. The transiently recruited lymphocytes adopt a specialized phenotype in the endometrium that regulates various placental physiological processes, including angiogenesis. Small non-coding RNAs, microRNAs (miRNAs) are emerging as principal bio-molecules regulating the development of lymphocytes and their angiogenic functions. However, no information is available in the context of endometrial lymphocytes in pregnancy. We hypothesize that miRNAs are involved in the development of endometrial lymphocytes and their angiogenic functions at the porcine maternal-fetal interface. Using a targeted Q-PCR approach for selected miRNAs involved in immune cell development, angiogenesis, and anti-angiogenesis, we conducted a study to screen endometrial lymphocytes associated with healthy and spontaneously arresting conceptus attachment sites (CAS) at two well-defined periods of fetal loss. Comparisons were made with endometrium and trophoblasts associated with healthy and arresting CAS. In addition, levels of putative mRNA targets and subsequent functional clustering of genes were studied in order to predict the biological mechanisms affected. We found several significant differences for miRNAs involved in immune cell development and angiogenesis (miR-296-5P, miR-150, miR-17P-5P, miR-18a, and miR-19a) between endometrial lymphocytes associated with healthy and arresting CAS. Significant differences were also found in endometrium and trophoblasts for some miRNAs (miR-20b, miR-17-5P, miR-18a, miR-15b-5P, and miR-222). Finally, selected mRNA targets showed differential expression in all groups. Our data, although associative, are the first to unravel the selected miRNAs involved in immune cell development and provide insights into their possible regulation in abortive pregnancy., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Laser capture microdissection (LCM) is an excellent and perhaps the only platform to isolate homogeneous cell populations from specific microscopic regions of heterogeneous tissue section, under direct microscopic visualization. The basic operations of the LCM system are based on (a) microscopic visualization of phenotypically identified cells of interest, (b) selective adherence of cells to a melting thermolabile film/membrane using a low-energy infrared laser (IR system) or photovolatization of cells within a selected region (UV system), (c) capturing or catapulting of structurally intact cells from a stained tissue section. RNA/DNA or protein can be extracted from the cell or tissue fragments for downstream applications to quantitatively study gene expression. This method can be applied to many downstream analyses including but not limited to quantitative real-time polymerase chain reaction (PCR), microarray, DNA genotyping, RNA transcript profiling, generation of cDNA library, mass spectrometry analysis, and proteomic discovery.The application of LCM is described here to specifically and reliably obtain a homogeneous cell population in order to extract RNA to study microRNA expression by quantitative real-time PCR.

Endometriosis is a chronic painful gynecological condition characterized by adherence and growth of endometrium outside of the uterine cavity. Neovascularization is essential to the developing endometriosis lesion to support its growth. Synuclein-γ (SNCG), a protein implicated in cellular proliferation, is associated with a broad range of malignancies as well as endometriosis. We hypothesized that SNCG plays an important role in the neovascularization and growth of endometriosis and blocking of SNCG will interfere with survival of endometriotic lesions in a mouse model. We developed SP012, a novel 12 amino acid peptide inhibitor of SNCG. SP012 inhibited three-dimensional endothelial cell tube formation in a dose-dependent manner. Using intravital microscopy, SP012 was shown to be successfully delivered to human endometriotic lesions in a xenograft mouse model in vivo. Alymphoid (BALB/c-Rag2-/-Il2rγ-/- lacking T, B and NK cells) mice were surgically induced with human endometriotic lesions and treated with SP012 or phosphate-buffered saline control. SP012 treated endometriotic lesions had decreased growth, development and vascularization at the time of necroscopy. Endometriotic lesions treated with SP012 also had fewer isolectin (+) microvessels. These results, using a mouse model, indicate that SNCG plays a role in the neovascularization and subsequent growth of human endometriotic lesions. Targeting SNCG function using peptide inhibitor might provide a potential therapeutic option for the treatment of endometriosis in the future., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

MicroRNAs (miRNAs) post-transcriptionally regulate a vast network of genes by inhibiting mRNA translation. Aberrant miRNA expression profiles have been implicated in pathologies and physiological processes including pregnancy and angiogenesis. Using our established model of implantation failure and spontaneous fetal loss in pigs (Sus scrofa), 236 miRNAs were profiled and compared between 1) non-pregnant and pregnant endometrium, 2) maternal and fetal tissues, and 3) viable and growth-arrested conceptus attachment sites by microarray and Real-Time PCR. Many significant differences in miRNA expression were observed between each of the aforementioned comparisons, and several were validated by PCR. Results indicated which miRNAs were important during pregnancy, which were elevated on the maternal or fetal side of the maternal-fetal interface, and they implicated the maternal expression of miR-10a, 27a, 29c, 323, 331-5p, 339-3p, 374b-5p, and 935 in the spontaneous loss observed in pigs. Several putative mRNA targets of the miRNAs (elevated in endometrium associated with arresting conceptuses) were assessed by quantitative Real-Time PCR and were depressed, supporting their regulation by miRNAs. Finally, targets were clustered by function to obtain ranked lists of gene networks that indicated which pathways/physiological processes might be important in non-pregnant (extracellular matrix factors) versus pregnant endometrium (nuclear transcription factor regulation), maternal (blood vessel development) versus fetal (neuronal differentiation) tissue, and healthy (extracellular matrix factors) versus arresting (GRAM domain) conceptus attachment sites. Overall, we demonstrate the presence of miRNAs on both sides of the maternal-fetal interface, implicate them in spontaneous fetal loss, and present a unique glimpse into the vast microRNAome of pregnancy.

Problem: Endometriosis affects 5-10% of women and is characterized by the growth of endometrial tissue outside of the uterus. Establishing new blood supply is a fundamental requirement for endometriosis lesion growth. Endothelial progenitor cells (EPCs), recruited by stromal cell-derived factor-1 (SDF-1), contribute to neoangiogenesis in endometriotic lesions. We hypothesized that SDF-1 is central to the neoangiogenesis and survival of endometriotic lesions, and blocking of SDF-1 will reduce vascularization of lesions in a mouse model., Method of Study: Using immunohistochemistry, we evaluated SDF-1 and CD34(+) EPCs in human endometriotic lesions and normal endometrium samples. EPCs were co-localized using CD34 and VEGFR2. Effects of SDF-1 blocking on endometriotic lesion survival were assessed in BALB/c-Rag2(-/-) /IL2rγ(-/-) mice engrafted with human endometrium and treated with SDF-1-blocking antibody or an isotype control. Weekly blood samples from experimental mice were analyzed for cytokines and EPCs., Results: SDF-1 and CD34(+) EPCs were abundant in human endometriotic lesions compared with eutopic endometrium. In our mouse model, SDF-1-blocking antibody reduced CD31(+) microvessels compared with isotype control., Conclusion: Blocking SDF-1 reduces neovascularization and survival of lesions in a mouse model of endometriosis., (© 2013 John Wiley & Sons Ltd.)

Endometriosis is a gynecological disease characterized by the growth of endometrium outside of the uterine cavity. It is often associated with dysmenorrhea, dyspareunia, pelvic pain and infertility. One of the key requirements for endometriotic lesions to survive is development of a blood supply to support their growth. Indeed, dense vascularization is characteristic feature of endometriotic lesions. This has led to the idea that suppression of blood vessel growth (anti-angiogenic therapy) may be a successful therapeutic approach for endometriosis. Potential effectiveness of anti-angiogenic therapies has been assessed in some animal models but there are no reports of human clinical trials. Without understanding the specific mechanism by which endometriosis lesions establish a new blood supply, short-term animal experiments will have limited value for translation into human medicine. Further, it is crucial to use appropriate animal models to assess efficacy of anti-angiogenic compounds. Syngeneic and autologous rodent models, where endometrial fragments are auto-transplanted into the peritoneal cavity are commonly used in anti-angiogenic therapy studies. Another approach is xenograft models where human endometrium is engrafted into immunodeficient mice. Here we review the animal models and experimental techniques used to evaluate anti-angiogenic therapies for endometriosis. We also review our own work on the role of stromal cell derived factor-1 in the recruitment of endothelial progenitor cells in endometriotic lesion angiogenesis, and the effects of the anti-angiogenic peptide ABT-898, a thrombospondin-1 mimetic, on endometriotic lesion growth and vascular development., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Endometriosis is a common cause of pelvic pain and infertility in women, and a common indication for hysterectomy, yet the disease remains poorly diagnosed and ineffectively treated. Because endometriotic lesions require new blood supply for survival, inhibiting angiogenesis could provide a novel therapeutic strategy. ABT-898 mimics the antiangiogenic properties of thrombospondin-1, so we hypothesized that ABT-898 will prevent neovascularization of human endometriotic lesions and that ABT-898 treatment will not affect reproductive outcomes in a mouse model. Endometriosis was induced in BALB/c-Rag2(-/-)Il2rg(-/-) mice by surgical implantation of human endometrial fragments in the peritoneal cavity. Mice received daily injections of ABT-898 for 21 days. Flow cytometry was performed to measure circulating endothelial progenitor cells in peripheral blood. Cytokines were measured in plasma samples. Half of the ABT-898-treated and control mice were euthanized to assess neovascularization of endometriotic lesions, using CD31(+) immunofluorescence. The remaining mice were mated and euthanized at gestation day 12. Endometriotic lesions increased circulating endothelial progenitor cells 13 days after engraftment, relative to baseline. Endometriotic lesions from ABT-898-treated mice exhibited reduced neovascularization, compared with controls, and lesions had fewer CD31(+) microvessels. Chronic treatment with ABT-898 did not lead to any fetal anomalies or affect litter size at gestation day 12, compared with controls. Our results suggest that ABT-898 inhibits neovascularization of human endometriotic lesions without affecting mouse fecundity., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Prenatal mortality is a prime concern for commercial swine industry in North America. Fetal losses occur throughout gestation but cluster in early (~day20) and mid (~day50) pregnancy. Adequate vascularization of the attachment site has emerged as a key factor contributing to fetal success. Since Insulin-Like Growth Factor (IGF) family members regulate angiogenesis in addition to promoting fetal development and growth, we hypothesized that conceptus success is governed by members of the IGF family. Using quantitative real time PCR, we analyzed expression of IGF family members (IGF-I, IGF-II, IGF-I Receptor (IGF-IR), IGF-IIR and their binding proteins, IGFBPs) in matched maternal and fetal tissues of healthy and arresting conceptuses at gestation days (gd) 20 and 50. IGF-II transcripts were 100 fold increased in both maternal and fetal tissues compared to IGF-I, but receptor transcripts were found in similar abundance irrespective of health status and gestation point. IGFBP3 was the most abundantly transcribed of the binding proteins. Using immunohistochemistry we confirmed the expression of IGF family members in maternal luminal and glandular epithelial cells, the endothelium of blood vessels and some scattered stromal cells. Our results suggest that IGF-I and II and their receptors are differentially expressed at the maternal and fetal components of the attachment site.

Successful pregnancy requires coordinated maternal-fetal cross-talk to establish vascular connections that support conceptus growth. In pigs, two waves of spontaneous fetal loss occur and 30-40% of conceptuses are lost before parturition. Previous studies associated these losses with decreased angiogenic and increased inflammatory cytokines. Chemokines, a sub-category of cytokines, and decoy receptors control leukocyte trafficking, angiogenesis and development. The availability of chemokines is regulated by three non-signalling decoy receptors: chemokine decoy receptor (D6), Duffy antigen receptor for chemokines (DARC) and Chemocentryx decoy receptor (CCX CKR). We hypothesized that the expression of these receptors and their chemokine ligands regulate the porcine pregnancy success or failure. Here, we describe for the first time the transcription and translation of all three decoy receptors and several chemokine ligands in endometrium and trophoblast associated with healthy and arresting conceptuses at gestation day (gd) 20 and gd50. Among decoy receptors, transcripts for DARC were significantly reduced in endometrium, whereas that for CCX CKR were significantly increased in endometrium and trophoblast at gd50 arresting compared with healthy sites. However, western blot analysis revealed no differences in decoy receptor expression between healthy and arresting tissues. Transcripts for decoy receptor ligands CCL2, CCL3, CCL4, CCL5, CCL11, CCL19, CCL21, CXCL2 and CXCL8 were stable between healthy and arresting littermates. Quantification by SearchLight chemiluminescent protein array confirmed ligand expression at the protein level. These data indicate that decoy receptors and ligands are expressed at the porcine maternal-fetal interface and dysregulation of decoy receptor (DARC and CCX CKR) transcripts occurs at sites of fetal arrest.

Background: Commercial swine breeds in North America undergo two waves of spontaneous fetal loss; one during peri-attachment and another during mid-gestation. Although an exact mechanism for this loss is not known, deficits in vasculature at the attachment sites appear to be a major cause. We hypothesized that a balance between pro-angiogenic and anti-angiogenic factors is needed at the maternal-fetal interface for successful conceptus development. Six selected members of the pro-angiogenic fibroblast growth factor (FGF) and platelet derived growth factor (PDGF) families and anti-angiogenic factor thrombospondin-1 (TSP-1) and its receptor CD36 were quantified and localized at the porcine maternal-fetal interface at early and midgestation time points., Methods: Mesometrial endometrium was collected from non-pregnant gilts (n = 8). Endometrial and chorioallantoic membrane samples were collected from healthy and arresting conceptus attachment sites at gestation day (gd) 20 (n = 8) and gd 50 (n = 8). At gd20 arresting conceptus attachment sites were distinguished by decreased vasculature of the placental membranes and decreased conceptus size. At gd50 arresting conceptuses attachment sites were identified by smaller conceptus length and weight measurements. Quantitative real time PCR was used to determine relative transcript levels of genes of interest, and cellular localization was determined by immunohistochemistry in paraffin embedded endometrial sections., Results: At gd20, endometrial samples from arresting conceptuses had elevated transcripts for bFGF, and PDGF-bb than healthy sites (p < 0.05). At gd50, bFGF, FGFR2, and CD36 were more abundant at arresting than at healthy conceptus attachment sites (p < 0.05). Chorioallantoic membrane from arresting conceptus attachment sites at gd20 had elevated transcripts for bFGF, FGFR1, FGFR2 and CD36 compared with healthy sites (p < 0.05). FGFR2 transcripts were more abundant in chorioallantoic membrane from arresting conceptuses at gd 50 (p < 0.05). Immunohistochemical localization of selected pro- and anti-angiogenic factors and receptors revealed their abundance in the luminal epithelium, uterine glands and perivascular areas of endometrium at gd20 and gd50., Conclusions: We provide comprehensive analysis of pro and anti-angiogenic factors at the porcine maternal fetal interface during early and mid-pregnancy. At mRNA levels, the majority of pro-angiogenic factors investigated were elevated at the sites of fetal arrest. These observations contrast with our previous findings of decreased Vascular Endothelial Growth Factor (VEGF) family members at arresting sites, and suggest that the bFGF family functions as a compensatory survival mechanism when major angiogenic proteins are decreasing at the sites of fetal arrest.

MicroRNAs comprise a family of small non-coding RNAs that modulate several developmental and physiological processes including pregnancy. Their ubiquitous presence is confirmed in mammals, worms, flies and plants. Although rapid advances have been made in microRNA research, information on stable reference genes for validation of microRNA expression is still lacking. Real time PCR is a widely used tool to quantify gene transcripts. An appropriate reference gene must be chosen to minimize experimental error in this system. A small difference in miRNA levels between experimental samples can be biologically meaningful as these entities can affect multiple targets in a pathway. This study examined the suitability of six commercially available reference genes (RNU1A, RNU5A, RNU6B, SNORD25, SCARNA17, and SNORA73A) in maternal-fetal tissues from healthy and spontaneously arresting/dying conceptuses from sows were separately analyzed at gestation day 20. Comparisons were also made with non-pregnant endometrial tissues from sows. Spontaneous fetal loss is a prime concern to the commercial pork industry. Our laboratory has previously identified deficits in vasculature development at maternal-fetal interface as one of the major participating causes of fetal loss. Using this well-established model, we have extended our studies to identify suitable microRNA reference genes. A methodical approach to assessing suitability was adopted using standard curve and melting curve analysis, PCR product sequencing, real time PCR expression in a panel of gestational tissues, and geNorm and NormFinder analysis. Our quantitative real time PCR analysis confirmed expression of all 6 reference genes in maternal and fetal tissues. All genes were uniformly expressed in tissues from healthy and spontaneously arresting conceptus attachment sites. Comparisons between tissue types (maternal/fetal/non-pregnant) revealed significant differences for RNU5A, RNU6B, SCARNA17, and SNORA73A expression. Based on our methodical assessment of all 6 reference genes, results suggest that RNU1A is the most stable reference gene for porcine pregnancy studies.

Commercial, North American pork breeds (Sus scrofa) experience significant loss of genetically-normal conceptuses during the peri-implantation (attachment) period and at mid-gestation (day 50 to 90 of the 114 day porcine gestation interval). Although exact causes for these losses are not defined, asynchronous in-utero development and deficits in vascularization of the endometrium and placenta appear to be involved. Understanding of normal maternal-fetal dialogue is critical to develop breeding or therapeutic strategies that improve fetal health and overall litter size in commercial pigs. The non-invasive, epitheliochorial porcine placenta permits investigation of maternal or fetal compartments without cross contaminating cells. We developed and use protocols to capture single, homogenous populations of porcine cells (endometrial lymphocytes, dendritic or endothelial cells) from histological sections using laser capture microdissection (LCM), a powerful tool for study of gene expression that reflects the in vivo environment. These data are compared with gene expression in biopsies of endometrium and of trophoblast from the same, attachment sites. Here we review justifications for selection of the genes we have studied and our published and in progress work. These data provide new insights into the roles of the endometrial immune environment in the regulation of the success and failure of porcine conceptuses.