Vasoactive intestinal peptide shapes first-trimester placenta trophoblast, vascular, and immune cell cooperation.

BACKGROUND AND PURPOSE: Extravillous trophoblast (EVT) cells are responsible for decidual stromal invasion, vascular transformation, and the recruitment and functional modulation of maternal leukocytes in the first-trimester pregnant uterus. An early disruption of EVT function leads to placental insufficiency underlying pregnancy complications such as preeclampsia and fetal growth restriction. Vasoactive intestinal peptide (VIP) is a vasodilating and immune modulatory factor synthesized by trophoblast cells. However, its role in first-trimester placenta has not been explored. Here, we tested the hypothesis that VIP is involved in first-trimester EVT outgrowth, spiral artery remodelling, balancing angiogenesis, and maintenance of immune homeostasis. EXPERIMENTAL APPROACH: First-trimester placental tissue (five to nine weeks of gestation) was collected, and was used for EVT outgrowth experiments, immunofluorescence, isolation of decidual natural killer (dNK) cells and decidual macrophages (dMA), and functional assays. Peripheral blood monocytes were differentiated with GM-CSF and used for angiogenesis assays. KEY RESULTS: In decidua basalis, VIP+ EVT were observed sprouting from cell columns and lining spiral arterioles. EVT migrating from placental explants were also VIP+. VIP increased EVT outgrowth and IL-10 release, whereas it decreased pro-inflammatory cytokine production in EVT, dNK cells, and dMA. VIP disrupted endothelial cell networks, both directly and indirectly via an effect on macrophages. CONCLUSION AND IMPLICATIONS: The results suggest that VIP assists the progress of EVT invasion and vessel remodelling in first-trimester placental bed in an immunologically "silent" milieu. The effects of VIP in the present ex vivo human placental model endorse its potential as a therapeutic candidate for deep placentation disorders.

Pomegranate Juice Supplementation Alters Utero-Placental Vascular Function and Fetal Growth in the eNOS-/- Mouse Model of Fetal Growth Restriction.

The eNOS-/- mouse provides a well-characterized model of fetal growth restriction (FGR) with altered uterine and umbilical artery function and reduced utero- and feto-placental blood flow. Pomegranate juice (PJ), which is rich in antioxidants and bioactive polyphenols, has been posited as a beneficial dietary supplement to promote cardiovascular health. We hypothesized that maternal supplementation with PJ will improve uterine and umbilical artery function and thereby enhance fetal growth in the eNOS-/- mouse model of FGR. Wild type (WT, C57Bl/6J) and eNOS-/- mice were supplemented from E12.5-18.5 with either PJ in their drinking water or water alone. At E18.5 uterine (UtA) and umbilical (UmbA) arteries were isolated for study of vascular function, fetuses and placentas were weighed and fetal biometric measurements taken. PJ supplementation significantly increased UtA basal tone (both genotypes) and enhanced phenylephrine-induced contraction in eNOS-/- but not WT mice. Conversely PJ significantly reduced UtA relaxation in response to both acetylcholine (Ach) and sodium nitroprusside (SNP), endothelium dependent and independent vasodilators respectively from WT but not eNOS-/- mice. UmbA sensitivity to U46619-mediated contraction was increased by PJ supplementation in WT mice; PJ enhanced contraction and relaxation of UmbA to Ach and SNP respectively in both genotypes. Contrary to our hypothesis, the changes in artery function induced by PJ were not associated with an increase in fetal weight. However, PJ supplementation reduced litter size and fetal abdominal and head circumference in both genotypes. Collectively the data do not support maternal PJ supplementation as a safe or effective treatment for FGR.

Melatonin Increases Fetal Weight in Wild-Type Mice but Not in Mouse Models of Fetal Growth Restriction.

Fetal growth restriction (FGR) presents with an increased risk of stillbirth and childhood and adulthood morbidity. Melatonin, a neurohormone and antioxidant, has been suggested as having therapeutic benefit in FGR. We tested the hypothesis that melatonin would increase fetal growth in two mouse models of FGR which together represent a spectrum of the placental phenotypes in this complication: namely the endothelial nitric oxide synthase knockout mouse (eNOS-/-) which presents with abnormal uteroplacental blood flow, and the placental specific Igf2 knockout mouse (P0+/-) which demonstrates aberrant placental morphology akin to human FGR. Melatonin (5 µg/ml) was administered via drinking water from embryonic day (E)12.5 in C57Bl/6J wild-type (WT), eNOS-/-, and P0+/- mice. Melatonin supplementation significantly increased fetal weight in WT, but not eNOS-/- or P0+/- mice at E18.5. Melatonin did, however, significantly increase abdominal circumference in P0+/- mice. Melatonin had no effect on placental weight in any group. Uterine arteries from eNOS-/- mice demonstrated aberrant function compared with WT but melatonin treatment did not affect uterine artery vascular reactivity in either of these genotypes. Umbilical arteries from melatonin treated P0+/- mice demonstrated increased relaxation in response to the nitric oxide donor SNP compared with control. The increased fetal weight in WT mice and abdominal circumference in P0+/-, together with the lack of any effect in eNOS-/-, suggest that the presence of eNOS is required for the growth promoting effects of melatonin. This study supports further work on the possibility of melatonin as a treatment for FGR.

Vitamin D, the placenta and early pregnancy: effects on trophoblast function.

Pregnancy is associated with significant changes in vitamin D metabolism, notably increased maternal serum levels of active vitamin D, 1,25-dihydroxyvitamin (1,25(OH)2D). This appears to be due primarily to increased renal activity of the enzyme 25-hydroxyvitamin D-1α-hydroxylase (CYP27B1) that catalyzes synthesis of 1,25(OH)2D, but CYP27B1 expression is also prominent in both the maternal decidua and fetal trophoblast components of the placenta. The precise function of placental synthesis of 1,25(OH)2D remains unclear, but is likely to involve localized tissue-specific responses with both decidua and trophoblast also expressing the vitamin D receptor (VDR) for 1,25(OH)2D. We have previously described immunomodulatory responses to 1,25(OH)2D by diverse populations of VDR-expressing cells within the decidua. The aim of the current review is to detail the role of vitamin D in pregnancy from a trophoblast perspective, with particular emphasis on the potential role of 1,25(OH)2D as a regulator of trophoblast invasion in early pregnancy. Vitamin D deficiency is common in pregnant women, and a wide range of studies have linked low vitamin D status to adverse events in pregnancy. To date, most of these studies have focused on adverse events later in pregnancy, but the current review will explore the potential impact of vitamin D on early pregnancy, and how this may influence implantation and miscarriage.

Vitamin D attenuates sphingosine-1-phosphate (S1P)-mediated inhibition of extravillous trophoblast migration.

INTRODUCTION: Failure of trophoblast invasion and remodelling of maternal blood vessels leads to the pregnancy complication pre-eclampsia (PE). In other systems, the sphingolipid, sphingosine-1-phosphate (S1P), controls cell migration therefore this study determined its effect on extravillous trophoblast (EVT) function. METHODS: A transwell migration system was used to assess the behaviour of three trophoblast cell lines, Swan-71, SGHPL-4, and JEG3, and primary human trophoblasts in the presence or absence of S1P, S1P pathway inhibitors and 1,25(OH)2D3. QPCR and immunolocalisation were used to demonstrate EVT S1P receptor expression. RESULTS: EVTs express S1P receptors 1, 2 and 3. S1P inhibited EVT migration. This effect was abolished in the presence of the specific S1PR2 inhibitor, JTE-013 (p < 0.05 versus S1P alone) whereas treatment with the S1R1/3 inhibitor, FTY720, had no effect. In other cell types S1PR2 is regulated by vitamin D; here we found that treatment with 1,25(OH)2D3 for 48 or 72 h reduces S1PR2 (4-fold; <0.05), but not R1 and R3, expression. Moreover, S1P did not inhibit the migration of cells exposed to 1,25(OH)2D3 (p < 0.05). DISCUSSION: This study demonstrates that although EVT express three S1P receptor isoforms, S1P predominantly signals through S1PR2/Gα12/13 to activate Rho and thereby acts as potent inhibitor of EVT migration. Importantly, expression of S1PR2, and therefore S1P function, can be down-regulated by vitamin D. Our data suggest that vitamin D deficiency, which is known to be associated with PE, may contribute to the impaired trophoblast migration that underlies this condition.

The atrial natriuretic peptide (ANP) knockout mouse does not exhibit the phenotypic features of pre-eclampsia or demonstrate fetal growth restriction.

The ANP knockout mouse is reported to exhibit pregnancy-associated hypertension, proteinuria and impaired placental trophoblast invasion and spiral artery remodeling, key features of pre-eclampsia (PE). We hypothesized that these mice may provide a relevant model of human PE with associated fetal growth restriction (FGR). Here, we investigated pregnancies of ANP wild type (ANP(+/+)), heterozygous (ANP(+/-)) and knockout (ANP(-/-)) mice. Maternal blood pressure did not differ between genotypes (E12.5, E17.5), and fetal weight (E18.5) was unaffected. Placental weight was greater in ANP(-/-) versus ANP(+/+) mice. Therefore, in our hands, the ANP model does not express phenotypic features of PE with FGR.

Amino acid composition of human uterine fluid: association with age, lifestyle and gynaecological pathology.

STUDY QUESTION: Do the amino acid levels of human uterine fluid vary with age, BMI, phase of menstrual cycle, benign pathology or diet? SUMMARY ANSWER: The levels of 18 amino acids in human uterine fluid were shown to be affected only by maternal diet. WHAT IS KNOWN ALREADY: Murine, bovine and ovine uterine amino acid content has been reported, but no reliable data on the human exist. Murine studies have demonstrated that the intrauterine periconceptional nutritional environment is affected by maternal diet. STUDY DESIGN, SIZE, DURATION: Uterine secretions were aspirated from 56 women aged 18-45 years. The women were recruited preoperatively from gynaecological theatre operating schedules or hysterosalpingo-contrast-sonography (HyCoSy) lists. A proportion of these women had proven fertility; however, the majority were being investigated for subfertility. The BMI, gynaecological history and dietary pattern of these women were also assessed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Reverse phase high performance liquid chromatography was used to analyse the concentrations of 18 amino acids within the uterine fluid and blood serum. The results were analysed against the women's stage of cycle, age, BMI and diet. MAIN RESULTS AND THE ROLE OF CHANCE: The profile of 18 amino acids in uterine fluid was described. In total, human uterine fluid was observed to contain an amino acid concentration of 3.54 mM (interquartile range: 2.27-6.24 mM). The relative concentrations of 18 amino acids were not significantly altered by age, BMI, cycle phase or the presence of specific benign gynaecological pathologies. However, a diet identified by a validated scoring system as being less healthy was associated with higher concentrations of asparagine (P = 0.018), histidine (P = 0.011), serine (P = 0.033), glutamine (P = 0.049), valine (P = 0.025), phenylalanine (P = 0.019), isoleucine (P = 0.025) and leucine (P = 0.043) in the uterine fluid compared with a healthier diet, defined as one with a higher intake of fresh vegetables, fruit, whole-grain products and fish and a low intake of red and processed meat and high fat dairy products. There were no significant correlations between serum amino acid concentrations and those in the uterine fluid. LIMITATIONS, REASONS FOR CAUTION: Our results enabled us to detect the effect of diet on the concentrations of amino acids in human uterine fluid; however, the study may not have had sufficient numbers to detect mild effects of BMI or age. WIDER IMPLICATIONS OF THE FINDINGS: These findings increase our understanding of the nutritional environment encountered by the preimplantation embryo, and indicate how periconceptional diet may alter this. Given the importance of early embryo environment for programming of development and future health, this information may aid in the development of nutritional interventions aimed at optimizing the preimplantation phase of human embryo development in vivo. STUDY FUNDING/COMPETING INTERESTS: This work was funded by the NIHR, the Medical Research Council (G0701153) and the University of Southampton and was supported by the NIHR BRC in Nutrition and Southampton University NHS Foundation Trust. The authors declare no conflicts of interest.