The study of progesterone action in human myometrial explants.

STUDY HYPOTHESIS: Myometrial explants represent a superior model compared with cell culture models for the study of human myometrial progesterone (P4) signalling in parturition. STUDY FINDING: Gene expression analysis showed myometrial explants closely resemble the in vivo condition and the anti-inflammatory action of P4 is not lost with labour onset. WHAT IS KNOWN ALREADY: Circulating P4 levels decline before the onset of parturition in most animals, but not in humans. This has led to the suggestion that there is a functional withdrawal of P4 action at the myometrial level prior to labour onset. However, to date, no evidence of a loss of P4 function has been provided, with studies hampered by a lack of a physiologically relevant model. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Myometrial biopsies obtained at Caesarean section were dissected into explants after a portion was immediately snap frozen (t = 0). Microarray analysis was used to compare gene expression of t = 0 with paired (i) explants, (ii) passage 4 myometrial cell cultures or (iii) the hTERT myometrial cell line. Western blotting and chemokine/cytokine assays were used to study P4 signalling in myometrial explants. MAIN RESULTS AND THE ROLE OF CHANCE: Gene expression comparison of t = 0 to the three models demonstrated that explants more closely resemble the in vivo status. At the protein level, explants maintain both P4 receptor (PR) and glucocorticoid receptor (GR) levels versus t = 0 whereas cells only maintain GR levels. Additionally, treatment with 1 µM P4 led to a reduction in interleukin-1 (IL-1) ß-driven cyclooxygenase-2 in explants but not in cells. P4 signalling in explants was PR-mediated and associated with a repression of p65 and c-Jun phosphorylation. Furthermore, the anti-inflammatory action of P4 was maintained after labour onset. LIMITATIONS/REASONS FOR CAUTION: There is evidence of basal inflammation in the myometrial explant model. WIDER IMPLICATIONS OF THE FINDINGS: Myometrial explants constitute a novel model to study P4 signalling in the myometrium and can be used to further elucidate the mechanisms of P4 action in human labour. LARGE SCALE DATA: Data deposited at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=gvmpggkurbgxfqf&acc=GSE77830. STUDY FUNDING AND COMPETING INTEREST: This work was supported by grants from the Joint Research Committee of the Westminster Medical School Research Trust, Borne (No. 1067412-7; a sub-charity of the Chelsea and Westminster Health Charity) and the Imperial NIHR Biomedical Research Centre. The views expressed are those of the author(s) and not necessarily those of the NHS or the Department of Health. The authors have no conflict of interest.

An evaluation of the ability of leucocyte depletion filters to remove components of amniotic fluid.

Haemorrhage remains an important cause of maternal mortality worldwide. Cell salvage carries a theoretical risk of amniotic fluid embolus syndrome and is too expensive for use in many parts of the world. To explore cheaper options, we investigated whether a leucocyte depletion filter alone removes components of pure amniotic fluid. Amniotic fluid was collected from 10 women during elective caesarean section and passed through a LeukoGuard® RS filter. Pre- and post-filtration samples were compared in the laboratory. Lamellar bodies and fetal squames were almost completely removed (filtration efficacy 96.6% and 99.9%, respectively; p<0.0001 and <0.0004), and hair was completely removed (p=0.002). Filtration had no effect on concentrations of α-fetoprotein, tissue factor or endothelin-1, or on the presence of meconium or vernix. Additional work is required to evaluate whether cell salvage using filtration alone may be useful in maternal haemorrhage in the developing world.

Prostaglandin F2-alpha receptor regulation in human uterine myocytes.

Investigations of the modulation of prostaglandin F(2alpha) receptor (FP) expression in primary cultures of human uterine myocytes showed that FP mRNA expression was reduced by progesterone, unaltered by cAMP (8-bromo cAMP or forskolin), but increased by the PKA antagonist H89. Interleukin (IL)-1beta, tumour necrosis factor-alpha and oxytocin increased FP mRNA expression and IL-6 and prostaglandin E(2) reduced FP mRNA expression. The changes in FP protein levels were similar to the mRNA responses. We found that the IL-1beta-induced increase in FP expression was mediated at least in part via protein kinase C (PKC), but was independent of mitogen-activated protein kinase, phospholipase C and PI3 kinase. Since IL-1beta activates NFkappaB, AP-1 and C/EBP, we over-expressed these transcription factors alone and in combination and found that only NFkappaB alone increased FP mRNA expression. Finally, we found that the IL-1beta-induced increase in FP expression was unaffected by progesterone and/or cAMP, but was accentuated by H89. These data suggest that the pregnancy-induced down-regulation in myometrial FP expression is mediated by progesterone and cAMP and that the increase with labour is induced by inflammatory cytokine activation of PKC and NFkappaB.

The mitogen-activated protein kinase dependent expression of prostaglandin H synthase-2 and interleukin-8 messenger ribonucleic acid by myometrial cells: the differential effect of stretch and interleukin-1{beta}.

Infection and uterine stretch are the common causes of preterm labor. IL-1beta plays a key role in infection-induced preterm labor and increases prostaglandin H synthase 2 (PGHS-2) and IL-8 expression. We have shown that mechanical stretch of uterine myocytes in vitro up-regulates the expression of PGHS-2 and IL-8. In this study, we tested the hypotheses that both IL-1beta and mechanical stretch increase the myometrial expression of PGHS-2 and IL-8 via MAPK activation and that their effects are synergistic. MAPK activation was assessed in myocytes obtained from pregnant women undergoing cesarean section before the onset of labor after exposure to IL-1beta and stretch either alone or in combination. Specific inhibitors of ERK, p38, and c-Jun N-terminal kinase were used to define the role of each in the increased expression of PGHS-2 and IL-8 mRNA. We found that both IL-1beta and stretch activated all three MAPK subtypes but that they had no synergistic effect. The inhibitor studies showed that stretch-induced increases in both PGHS-2 and IL-8 mRNA expression were ERK1/2 and p38 dependent and that IL-1beta-induced increases of PGHS-2 mRNA expression were also ERK1/2 and p38 dependent, but those of IL-8 were dependent only on ERK1/2 activation. These data show that exposure of human uterine myocytes to both stretch and IL-1beta activates the MAPK system, which is responsible for the increase in PGHS-2 and IL-8 mRNA expression. We found no evidence of a synergistic effect of IL-1beta and stretch on myometrial expression of PGHS-2 and IL-8 mRNA.

Flow cytometric characterisation of cells of differing densities isolated from human term placentae and enrichment of villous trophoblast cells.

Cells were isolated from human term placentae by trypsinisation of fragments of chorionic villi and fractionation of cells on a Percoll density gradient into six layers. A panel of 10 monoclonal antibodies to antigens on or in trophoblast cells (placental alkaline phosphatase (PLAP), cytokeratin-7, beta-human chorionic gonadotrophin (beta-hCG), human leucocyte antigen-G (HLA-G)), leucocytes (CD45), monocytes, macrophages, dendritic cells, B cells (HLA class II), mesenchyme cells (vimentin), fibroblasts (fibroblast antigen) and nucleated cells excluding villous trophoblast (HLA class I, CD9) was used to characterise the cells by flow cytometry. For staining intracellular antigens (cytokeratin, vimentin, beta-hCG) the cells were first fixed and permeabilised. The upper two layers from the gradient (density 1.013-1.039 g/ml) contained predominantly PLAP-positive cells or fragments, probably derived from the syncytiotrophoblast. Cytokeratin-positive cells accumulated mainly in the layer of density 1.039-1.052 g/ml and comprised the majority of the cell types identified in this fraction. Few or no cells reactive with antibodies to beta-hCG or HLA-G were identified in any layer. Non-trophoblast cells were heavier, being present mainly at densities 1.052-1.079 g/ml (CD45, HLA class I, vimentin) and 1.066-1.092 g/ml (fibroblast). Fewer than 10% of cells in any layer were HLA class II- or CD9-positive. Further purification of trophoblast cells was by negative immunomagnetic separation with removal of CD45-positive cells and HLA class II-positive cells to less than 1%. On culture of the cells from each layer, those of density 1.039-1.066 g/ml exhibited characteristics of cytotrophoblast cells; they secreted high levels of human chorionic gonadotrophin and formed adherent multinucleate cells. This procedure enabled the selection and enrichment of cytotrophoblast cells and/or syncytiotrophoblast fragments that are suitable for cellular and molecular studies.

A longitudinal study of resting peripheral blood flow in normal pregnancy and pregnancies complicated by chronic hypertension and pre-eclampsia.

OBJECTIVES: To investigate the hypothesis that reduced resting tissue blood flow precedes the clinical onset of pre-eclampsia in women at risk of the disease. METHODS: We used venous occlusion plethysmography to compare resting calf muscle blood flow in 18 normal pregnant controls, 18 pregnant women with chronic hypertension, and 23 pregnant women at increased risk of developing pre-eclampsia. Calf blood flow was measured at 16, 20, 24, 28, 32 and 36 weeks of gestation. RESULTS: Blood flow increased with gestation in normal pregnancy (P = 0.004) and chronic hypertension (P = 0.006), but not in the 'at risk' women who did not develop pre-eclampsia (P = 0.36). In contrast, blood flow decreased significantly in eight out of the 23 women 'at risk', who developed pre-eclampsia (P < 0.00001, ANOVA). The decrease in flow preceded the clinical diagnosis of the pre-eclampsia by several weeks. Moreover, a significant inverse correlation was observed between resting blood flow and plasma uric acid concentrations (r = -0.86, P = 0.03) in the women that developed pre-eclampsia. CONCLUSIONS: We have shown that reduced resting blood flow precedes the clinical onset of pre-eclampsia independently of hypertension per se. These findings support the notion that impaired tissue blood flow may be involved at an early stage in the pathophysiology of the disease.

Progesterone and the Repression of Myometrial Inflammation: The Roles of MKP-1 and the AP-1 System.

Progesterone (P4) maintains uterine quiescence during pregnancy and its functional withdrawal is associated with increased prostaglandin synthesis and the onset of labor. In primary human myometrial cells, the glucocorticoid receptor (GR) rather than the P4 receptor mediates P4 antagonism of IL-1ß-induced cyclooxygenase-2 (COX-2) expression, the rate-limiting enzyme in prostaglandin synthesis. We now report that P4 also acts via GR to induce MAPK phosphatase (MKP)-1 and knockdown of MKP-1 impairs the ability of P4 to repress IL-1ß-dependent COX-2 induction. Microarray analysis revealed that P4 repressed preferentially activator protein-1-responsive genes in response to IL-1ß. Consistent with these observations, we found that the ability of P4 to reduce c-Jun activation was lost upon GR as well as MKP-1 knockdown. Interestingly, c-Jun levels in human myometrial cells declined upon GR and MKP-1 knockdown, which suggests the presence of an activator protein-1 feedback loop. This is supported by our observation that c-Jun levels declined after an initial rise in primary myometrial cells treated with phorbol 12-myrisatate 13-acetate, a potent activator of c-Jun N-terminal kinase. Finally, we show that MKP-1 is an intermediate in P4-mediated repression of some but not all IL-1ß-responsive genes. For example, P4 repression of IL11 and IRAK3 was maintained upon MKP-1 knockdown. Taken together, the data show that P4 acts via GR to drive MKP-1 expression, which in turn inhibits IL-1ß-dependent c-Jun activation and COX-2 expression.

Placental regulation of insulin-like growth factor axis in monochorionic twins with chronic twin-twin transfusion syndrome.

To test the hypothesis that severe growth restriction (intrauterine growth retardation) in donor twins with chronic twin-twin transfusion syndrome (TTTS), a common complication of monochorionic twin pregnancy, is due to an aberration in the insulin-like growth factor (IGF) axis, we studied 25 sets of monochorionic twins with (n = 13) and without (n = 12) TTTS. Maternal and cord blood samples were collected at birth and analyzed for IGF-I, IGF-II, IGF-binding protein-1 (IGFBP-1), and IGFBP-1 phosphorylation status. Fetal IGF-II levels in the recipient twins with TTTS were higher than those in the donor twins (829 +/- 45 vs. 543 +/- 60 ng/mL; P < 0.001), but were comparable with those in the non-TTTS twin pairs. IGF-I levels in recipient and donor twin pairs were similar. The total IGFBP-1 concentration was higher in the donor twins than in the recipients (1153 +/- 296 vs. 419 +/- 108 ng/mL; P < 0.001) and non-TTTS twin pairs (P < 0.01). The percent less phosphorylated IGFBP-1 was higher in the recipients than in the donor twins (P < 0.05). There were no differences in IGF-I, IGF-II, and IGFBP-1 levels between non-TTTS twin pairs. Maternal levels of IGFs were comparable in the two groups. In the TTTS group, fetal birth weight gave a positive correlation with serum IGF-II levels (y = 0.25x + 361.1; r = 0.47; P < 0.05), and a negative association with IGFBP-1 levels (y = -0.72x + 1593.6; r = 0.58; P < 0.01). Our data argue against intertwin transfusion as the cause of intrauterine growth retardation in the donor twin and provide evidence that the placenta is the key regulator of the fetal IGF axis, especially when fetal genotype and maternal environments are similar.

Superfused microcarrier cultures of choriocarcinoma cells: a dynamic model for studying transport of glucose and amino acids.

We here describe a novel procedure for culturing BeWo and JAr choriocarcinoma cells on solid microcarrier beads. The regime developed to stir the beads resulted, after about 10 days, in small aggregates of two to six beads covered with a layer of differentiated cells. Bead aggregates were packed into small columns and superfused, providing a dynamic in vitro system for studying the uptake mechanisms for sugars and amino acids. The rapid, unidirectional uptake of tritiated L-phenylalanine, L-serine, L-arginine and D-glucose was determined, relative to an extracellular reference tracer, in cells superfused in 0.5 ml (range 0.3-0.8 ml) columns. Several sequential measurements could be made in the same column. Twenty-four hour pre-incubation with dexamethasone (0.25 microM) was found to increase the transport of D-glucose. Uptake of D-glucose was reduced by over 80 per cent following 20 min perfusion of the cells with 1 mM phloretin. Pre-incubation with growth hormone (0.2 microgram/ml) decreased the transport of serine, whereas nicotine (0.5 microgram/ml) decreased both serine and phenylalanine uptake. Atropine (1 mM) or 5-oxoproline (0.5 mM) had no short-term effects on amino acid uptake. Insulin (12.5 mIU/ml) had no effect on the transport of the amino acids but caused a small but significant increase in glucose transport (P < 0.05). This model allows characterization of human trophoblast function without the complications resulting from the presence of other cell types in placental slices or fragments.

Regulation of the choline transport system in superfused microcarrier cultures of BeWo cells.

BeWo choriocarcinoma cells were cultured onto solid microcarrier beads, packed into syringe barrels and superfused. The unidirectional choline uptake across the microvillous membrane of the cells was measured by a rapid single-circulation paired-tracer dilution procedure using methyl[3H]choline with D-[14C]mannitol as the extracellular reference molecule. Choline influx was saturable with a K(t) of 214+/-15 microM and a V(max) of 45.29+/-0.94 nmol/min/mg of cell protein. Uptake of labelled choline was partially inhibited by nicotine, strongly inhibited by hemicolinium-3, and was reduced by about 50 per cent in sodium-free perfusates. A range of agents was added to the stirrer flasks 24 h prior to the experiments to determine if intracellular or extracellular levels of choline or its metabolic product, acetylcholine, regulated choline uptake. Pre-incubation with 2 mM choline reduced the choline maximal uptake by half, while pre-incubation with 100 microM alpha-NETA [2-(alpha-naphthoyl)ethyltrimethyl-ammonium] reduced the influx by 77 per cent. Choline influx was also reduced to about half in the presence of 100 microM vesamicol, bethanecol or neostigmine. It is concluded that BeWo cells possess a choline transporter similar to that described in isolated cytotrophoblasts and syncytiotrophoblast microvillous membrane preparations, and that uptake appeared to be regulated by both intracellular and extracellular concentrations of choline and acetylcholine. Therefore, these cells provide a novel model for studying the role of acetylcholine in human placenta.

Transport of large neutral amino acids into BeWo cells.

BeWo choriocarcinoma cells were cultured onto solid microcarrier beads, packed into columns and superfused. Unidirectional influx of l -phenylalanine (l -phe) and l -leucine (l -leu) across the microvillous border of the cells was studied using a rapid paired-tracer dilution technique. Influx of l -phe and l -leu comprised both saturable and non-saturable components. K(m)values for l -phe and l -leu were 0.57+/-0.01 m m and 0.05+/-0.01 m m, respectively, with V(max)values of 120.4+/-0.5 nmol/mg/min and 41. 7+/-0.2 nmol/mg/min. Non-saturable uptake components were 29.0+/-0.1 nmol/mg/m m and 37.9+/-0.1 nmol/mg/min/m m respectively. l -leu uptake was found to be sodium-independent. The uptake of l -[(3)H]phe was strongly inhibited (90-100 per cent) by unlabelled l -phe, d -phe, l -leu or 2-aminoendobicyclo-[2,2, 1]-heptane-2-carboxylic acid (BCH) but not by l -arginine (l -arg) or methyl alpha-aminoisobutric acid (Me-AIB). Pre-incubation of Bewo cultures for 24 h in the presence of an additional 1.2 m ml -phe (simulating maternal phenylketonuria) significantly reduced both the K(m)and V(max)components of l -phe influx. l -arg (2 m m) had no effect on l -leu influx whereas 2 m ml -phe completely inhibited saturable l -leu influx. These data suggest that the microvillous border of differentiated BeWo cells transport large neutral amino acids predominantly via system L rather than by B(0) or y(+)L transporters.

Identification and estimation of cell types in mixed primary cell cultures of early and term human placenta.

Placental villi of early and term human placentae were dissociated with trypsin and mixed cell cultures were established. The different cell types were identified and estimated over a 14-day culture period using antibodies to keratin and vimentin filaments and their capacity to phagocytose yeast. The three main cell types were found to be epithelial-, macrophage- and fibroblast-like cells. The epithelial-like cells can be further divided into the multinucleated and the small- and medium-sized round cells, and these are most likely to be derived from the trophoblast. The cellular composition of cultures were different for early and term placentae and also varied characteristically over the 14-day cultures period.

Atrial natriuretic peptide mediated polyuria: pathogenesis of polyhydramnios in the recipient twin of twin-twin transfusion syndrome.

We studied the role of atrial natriuretic peptide (ANP) in the pathophysiology of polyhydramnios in monochorionic (MC) twins with and without twin-twin transfusion syndrome (TTTS). Matched maternal, fetal blood samples and amniotic fluids (AF) were obtained in utero (n=12) and at birth (n=20) from MC twins with TTTS. Blood and amniotic fluid samples were also collected from non-TTTS MC twin pairs in utero (n=6) and at birth (n=20). In both groups cellular localization of ANP in the fetal kidney and heart was performed using anti ANP rabbit polyclonal antibody. Concentrations of ANP in pg/ml were determined by radioimmunoassay.In recipient fetuses, ANP levels were higher than the donors both in utero (P< 0.001) and at birth (P< 0.001). No such differences were found between the non-TTTS twins. In the TTTS group maternal ANP levels were lower than the non-TTTS group (P< 0.05). A linear relationship was found between fetal ANP levels and the AF volumes removed at fetal blood sampling (r(2)=0.68;P< 0.01, n=12). ANP was localized predominantly to the cytoplasm of the distal convoluted tubules of the fetal kidney and heart, and the intensity of immunostaining for ANP in kidney and heart were markedly greater in the recipient than the donor twin. No such differences were found between the twin pairs. These data suggest that polyhdramnios in the recipient twin occurs as a consequence of ANP mediated increase in fetal urine output and raises the possibility of direct fetal therapy with ANP blocking agents.

Discordant fetal leptin levels in monochorionic twins with chronic midtrimester twin-twin transfusion syndrome.

The objective of this study was to determine the plasma leptin concentrations in monochorionic twin fetuses with and without twin-twin transfusion syndrome (TTTS). Paired maternal and fetal blood samples were obtained at birth from monochorionic twin pregnancies complicated with (n=12) or without TTTS (n=12). Amniotic fluid samples were also collected from twin pairs at amnioreduction and/or fetal blood sampling in utero. Plasma and amniotic fluid leptin concentrations were measured by radio-immunoassay. Fetal leptin levels in the growth-restricted donor were lower than the recipient twin of the TTTS group (Delta mean 3.7; CI 2.6 to 4.7 ng/ml; P< 0.001). Fetal leptin levels were comparable between non-TTTS twin pairs (Delta mean 0.9; CI 0.1 to 1.4 ng/ml; P=0.10) and recipient twins of TTTS (P=NS). Maternal plasma concentrations of leptin were comparable between the two groups and were higher than the fetal levels. There was a positive association between cord leptin levels and birthweight of twin pairs (y=0.002x-0.37; r=0.58; P< 0.01; n=48). A significant positive relation was also found between delta leptin levels and percentage discordance in birthweight in the TTTS group (y=0.25x-2.21; r=0.82; P< 0.001, n=12). In conclusion, leptin levels in the recipient twins were three times higher than their growth restricted donor twins. However further studies are warranted to elucidate the underlying mechanism.

Free amino acid concentrations in human first and third trimester placental villi.

Homogenates of first (10.1 +/- 1.0 weeks) and third trimester placental villi were analysed for free amino acid concentrations. As has been previously reported, several amino acids showed increased concentrations during early pregnancy when compared to term. In addition, marked differences were seen in the levels of ethanolamine (which was increased fivefold in term placentae) and phosphoethanolamine (which was decreased by almost 97 per cent of the value measured at 10 weeks gestation). The implications of these results are discussed.

Prospective function of placental leptin at maternal-fetal interface.

Leptin is an endocrine and a growth factor which is important for regulation of body fat, feeding, and energy homeostasis. The anti-obesity function of leptin has been recently extended to reproduction, puberty and pregnancy as an endocrine signal to the hypothalamus. Leptin controls the functional integrity of the feto-placental unit thereby maintaining pregnancy by virtue of its immunomodulatory property via T lymphocytes or other proto-oncogenes. Dysregulation of autocrine/paracrine function of leptin at feto-placento-maternal interface may be implicated in the pathogenesis of recurrent miscarriage gestational diabetes, pre-eclampsia and intra-uterine fetal growth retardation including disturbance of fetal bone turnover. This review will focus on the role of leptin in normal and abnormal pregnancy and fetal growth.

Nitric oxide synthase activities in human myometrium and villous trophoblast throughout pregnancy.

OBJECTIVE: To study the changes in nitric oxide synthase activities in human myometrium and trophoblast throughout pregnancy and around delivery. METHODS: Samples of villous trophoblast were collected from women undergoing elective cesarean delivery at term (n = 12) or voluntary termination of pregnancy in the first (n = 27) or second (n = 11) trimesters of pregnancy. Myometrial samples were obtained from nonpregnant women undergoing hysterectomy (n = 5) and pregnant women both before (n = 7) and after (n = 7) the onset of spontaneous labor at term. Nitric oxide synthase activity was quantified for homogenized samples using the L-citrulline assay in the presence and absence of calcium. RESULTS: The highest levels of nitric oxide synthase activity were found in first-trimester villi (range 2-29 nmol L-citrulline/minute/g protein), with a significant fall in activity in the third trimester (range 2-10 nmol L-citrulline/minute/g protein; P < .001 for both calcium-dependent and calcium-independent activity). Myometrial activities were relatively low compared with those in the trophoblast (0-2 nmol L-citrulline/minute/g protein), with no significant differences in calcium-dependent activities between subgroups. Myometrial calcium-independent activities were lower in pregnant than in nonpregnant women (P = .007), with those in labor having levels higher than those not in labor (P = .048). CONCLUSION: Levels of nitric oxide synthase activity are relatively high in villous trophoblast, particularly during the first trimester. Although the contribution to total nitric oxide production in the uterus by myometrial nitric oxide synthase appears to be relatively small, nitric oxide produced by the trophoblast may play a role in maintaining uterine quiescence by a paracrine effect. Further work is needed to test this hypothesis and explore other possible roles for trophoblast-derived nitric oxide in early pregnancy.

Potent activation of nitric oxide synthase by garlic: a basis for its therapeutic applications.

Garlic (Allium sativum L.) is thought to have a variety of therapeutic applications including inhibition of platelet aggregation. Many of the therapeutic actions of garlic parallel the physiological effects of nitric oxide and may be explained by its ability to increase nitric oxide synthase activity intracellularly. Our studies showed that both water and alcoholic extracts of garlic are very potent inhibitors of platelet aggregation induced by epinephrine and ADP. Similar dilutions of garlic extract also activated nitric oxide synthase activity in isolated platelets in vitro. The same extract was also very effective in activating nitric oxide synthase activity in placental villous tissue. The addition of garlic extracts increased nitric oxide synthase activity in a dose-dependent manner. Nitrite levels in the supernatants of incubated placental villous tissue were similarly increased. Activation of calcium-dependent nitric oxide synthase and the subsequent production of nitric oxide is probably the most novel mechanism yet claimed by which garlic can exert its therapeutic properties.

Placental nitric oxide metabolism.

There is increasing evidence that nitric oxide (NO) has a role in pregnancy. NO is synthesized from L-arginine by NO synthase (NOS), which can exist either as a calcium-dependent or a calcium-independent isoform of the enzyme. Both isoforms are present in placental villi and the authors have measured NOS activities in tissues from early and term normal, pre-eclamptic and growth-retarded pregnancies. Higher activities were seen in first trimester placental villi than at term. An impairment of NO metabolism occurred in placental villi from pre-eclamptic and growth-retarded pregnancies. Smoking also results in decreased NOS activities in the placental villi, suggesting that problems attributed to smoking during pregnancy could be linked to NO metabolism. Polyamines arginine and citrulline (all of which are important metabolites in the NO pathway) were also measured in placental villous tissues. The data presented in this review article are from work carried out in the authors' laboratories and suggest that alterations in the placental arginine-NO pathway may not only play a role in the physiological changes of advancing gestation but may also contribute to the pathophysiology of pre-eclampsia and fetal growth retardation.

Preparation of human trophoblast cells for culture.

The difficulty in obtaining a preparation of pure trophoblast cells for culture can be appreciated by understanding the structure of the placenta. The outer surface of the chorionic villi is covered by the syncytiotrophoblast, underlying which is a single cell layer of cytotrophoblast cells that sits on the basement membrane. Microvascular vessels connect this cell layer to the umbilical arteries and vein. The apical membrane of the syncytiotrophoblast is folded into numerous microvilli, and this layer forms a syncytium. Disaggregation of this villous tissue results in a broken syncytial membrane, releasing not only the cytotrophoblastic cells, but also the rest of the villous cell population (i.e., Hofbauer cells or macrophages, fibroblasts, grant cells, some adhering decidual and endothelial cells) as well as DNA from the nuclei of the syncytium. Separation of trophoblast from this heterogeneous cell population has proven to be a challenge.