Fetoplacental Growth Restriction Research Articles

Restoring Angiotensin Type 2 Receptor Function Reverses PFOS-Induced Vascular Hyper-Reactivity and Hypertension in Pregnancy.

Perfluorooctane sulfonic acid (PFOS) exposure during pregnancy induces hypertension with decreased vasodilatory angiotensin type-2 receptor (AT2R) expression and impaired vascular reactivity and fetal weights. We hypothesized that AT2R activation restores the AT1R/AT2R balance and reverses gestational hypertension by improving vascular mechanisms. Pregnant Sprague-Dawley rats were exposed to PFOS through drinking water (50 μg/mL) from gestation day (GD) 4-20. Controls received drinking water with no detectable PFOS. Control and PFOS-exposed rats were treated with AT2R agonist Compound 21 (C21; 0.3 mg/kg/day, SC) from GD 15-20. In PFOS dams, blood pressure was higher, blood flow in the uterine artery was reduced, and C21 reversed these to control levels. C21 mitigated the heightened contraction response to Ang II and enhanced endothelium-dependent vasorelaxation in uterine arteries of PFOS dams. The observed vascular effects of C21 were correlated with reduced AT1R levels and increased AT2R and eNOS protein levels. C21 also increased plasma bradykinin production in PFOS dams and attenuated the fetoplacental growth restriction. These data suggest that C21 improves the PFOS-induced maternal vascular dysfunction and blood flow to the fetoplacental unit, providing preclinical evidence to support that AT2R activation may be an important target for preventing or treating PFOS-induced adverse maternal and fetal outcomes.

Activation of angiotensin type 2 receptor attenuates testosterone-induced hypertension and uterine vascular resistance in pregnant rats†.

Preeclampsia is a pregnancy-related hypertensive disorder with unclear mechanisms. While hypersensitivity to angiotensin II via vasoconstrictive angiotensin type-1 receptor (AT1R) is observed in preeclampsia, the importance of vasodilatory angiotensin type-2 receptor (AT2R) in the control of vascular dysfunction is less clear. We assessed whether AT1R, AT2R, and endothelial nitric oxide synthase (eNOS) expression are altered in placental vessels of preeclamptic women and tested if ex vivo incubation with AT2R agonist Compound 21 (C21; 1μM) could restore AT1R, AT2R, and eNOS balance. Further, using a rat model of gestational hypertension induced by elevated testosterone, we examined whether C21 (1μg/kg/day, oral) could preserve AT1R and AT2R balance and improve blood pressure, uterine artery blood flow, and vascular function. Western blots revealed that AT1R protein level was higher while AT2R and eNOS protein were reduced in preeclamptic placental vessels, and AT2R agonist C21 decreased AT1R and increased AT2R and eNOS protein levels in preeclamptic vessels. In testosterone dams, blood pressure was higher, and uterine artery blood flow was reduced, and C21 treatment reversed these levels similar to those in controls dams. C21 attenuated the exaggerated Ang II contraction and improved endothelium-dependent vasorelaxation in uterine arteries of testosterone dams. These C21-mediated vascular effects were associated with decreased AT1R and increased AT2R and eNOS protein levels. C21 also increased serum nitrate/nitrite and bradykinin production in testosterone dams and attenuated the fetoplacental growth restriction. Thus, AT1R upregulation and AT2R downregulation are observed in preeclampsia and testosterone model, and increasing AT2R activity could help restore AT1R and AT2R balance and improve gestational vascular function.

Testosterone Decreases Placental Mitochondrial Content and Cellular Bioenergetics.

Placental mitochondrial dysfunction plays a central role in the pathogenesis of preeclampsia. Since preeclampsia is a hyperandrogenic state, we hypothesized that elevated maternal testosterone levels induce damage to placental mitochondria and decrease bioenergetic profiles. To test this hypothesis, pregnant Sprague–Dawley rats were injected with vehicle or testosterone propionate (0.5 mg/kg/day) from gestation day (GD) 15 to 19. On GD20, the placentas were isolated to assess mitochondrial structure, copy number, ATP/ADP ratio, and biogenesis (Pgc-1α and Nrf1). In addition, in vitro cultures of human trophoblasts (HTR-8/SVneo) were treated with dihydrotestosterone (0.3, 1.0, and 3.0 nM), and bioenergetic profiles using seahorse analyzer were assessed. Testosterone exposure in pregnant rats led to a 2-fold increase in plasma testosterone levels with an associated decrease in placental and fetal weights compared with controls. Elevated maternal testosterone levels induced structural damage to the placental mitochondria and decreased mitochondrial copy number. The ATP/ADP ratio was reduced with a parallel decrease in the mRNA and protein expression of Pgc-1α and Nrf1 in the placenta of testosterone-treated rats compared with controls. In cultured trophoblasts, dihydrotestosterone decreased the mitochondrial copy number and reduced PGC-1α, NRF1 mRNA, and protein levels without altering the expression of mitochondrial fission/fusion genes. Dihydrotestosterone exposure induced significant mitochondrial energy deficits with a dose-dependent decrease in basal respiration, ATP-linked respiration, maximal respiration, and spare respiratory capacity. In summary, our study suggests that the placental mitochondrial dysfunction induced by elevated maternal testosterone might be a potential mechanism linking preeclampsia to feto-placental growth restriction.

Maternal hypertension and feto-placental growth restriction is reversed by sildenafil: Evidence of independent effects of circulating nitric oxide levels

Sildenafil has shown nitric oxide (NO)-independent pleiotropic effects, however the mechanisms involved are unclear. We investigated the protective effects of sildenafil against hypertension in pregnancy and feto-placental growth restriction induced by NO inhibition, and if sodium nitrite-derived NO formation influences sildenafil effects. We evaluated the plasmatic levels of NO metabolites, cyclic guanosine monophosphate (cGMP), oxidative stress and myeloperoxidase, which are involved in endothelial dysfunction during hypertension in pregnancy. Also, we performed in vitro experiments to examine cell viability and NO synthesis in human umbilical vein endothelial cells (HUVECs) cultures incubated with plasma from healthy or hypertensive pregnant rats treated (or not) with both drugs, either alone or in association. Sildenafil blunted hypertension in pregnancy and protected against feto-placental growth restriction induced by NO inhibition and these effects of sildenafil alone were similar to those presented by its association with sodium nitrite. Protective effects of sildenafil were observed even with low plasmatic NO levels and were not followed by increases in cGMP levels. Also, sildenafil, but not sodium nitrite, blunted the increases in myeloperoxidase activity. Both drugs (isolated or in association) presented antioxidant effects. Plasma from hypertensive pregnant rats treated with sildenafil, but not sodium nitrite alone, increased the viability of HUVECs. NO synthesis in HUVECs cultures was increased with plasma from rats treated with both drugs. We conclude that sildenafil effects are not dependent of circulating NO levels in hypertension and feto-placental growth restriction. These findings may reflect a protection against myeloperoxidase and pro-oxidant activation in hypertension in pregnancy.

Kiss1 and Kiss1r mRNA expression in the rat placenta: Changes with gestational age and regulation by glucocorticoids

IntroductionKisspeptin, the neuropeptide product of the KISS1 gene, is synthesized by neurons within the hypothalamus and is critical for fertility. Human placenta also expresses KISS1 and kisspeptin receptor (KISS1R) mRNA within the trophoblast compartment, where it is thought to act as a physiological invasion inhibitor. MethodsWe determined the expression of Kiss1 mRNA in rat placenta and examined the effect of gestational age and feto-placental growth restriction, achieved through excess maternal glucocorticoid exposure. ResultsDexamethasone induced fetal growth restriction at both day 16 and day 22 of gestation, but placental growth restriction only at day 22. Real-time quantitative RT-PCR revealed an increase in Kiss1 and Kiss1r mRNA from day 16–22 in the labyrinth and junctional zones of the rat placenta. Immunolocalization confirmed kisspeptin expression in the placenta and was prominent in trophoblast tissue. Dexamethasone exposure elevated the expression of Kiss1 mRNA in the labyrinth and junctional zones of day 16 placentas. In contrast, Kiss1 mRNA in the labyrinth zone was reduced following dexamethasone-treatment at day 22. Kiss1r expression was increased in both placental zones at day 16 and 22 in response to dexamethasone-treatment. ConclusionsWe confirm the presence of Kiss1 and Kiss1r mRNA in the rat placenta with expression increasing over the final third of pregnancy, suggestive of a role in restricting placental growth. Furthermore, the effects of dexamethasone on placental Kiss1/Kiss1r suggest glucocorticoid-induced placental growth retardation could be mediated, in part, via early stimulation of Kiss1 and the subsequent inhibition of trophoblast proliferation and invasion.

Adrenomedullin 2/Intermedin Regulates HLA-G in Human Trophoblasts1

Adrenomedullin 2 (ADM2), also referred to as intermedin (IMD), is expressed in trophoblast cells in human placenta and enhances the invasion and migration of first-trimester HTR-8SV/neo cells. Further infusion of ADM2 antagonist in pregnant rat causes fetoplacental growth restriction, suggesting a role for ADM2 in maintaining a successful pregnancy. This study was undertaken to assess whether ADM2 protein is present in decidual tissue and colocalized with HLA-G-positive cytotrophoblast cells and natural killer cells; to assess whether ADM2 regulates expression of HLA-G in trophoblast cells; and to identify whether mitogen-activated protein kinase (MAPK) signaling pathway is involved in ADM2-induced trophoblast cell invasion and migration. Using immunohistochemical methods and RT-PCR, this study shows that ADM2 protein is colocalized with HLA-G-expressing cytotrophoblast cells as well as with NCAM1 (CD56) immunoreactivity in human first-trimester decidual tissue, and that ADM2 mRNA is expressed in peripheral blood natural killer cells. Further, ADM2 dose dependently increases the expression of HLA-G antigen in HTR-8SV/neo cells as well as in term placental villi explants, suggesting involvement of ADM2 in the regulation of HLA-G in trophoblast cells. In addition, interference with the activity of RAF and MAPK3/1 by their inhibitors, manumycin and U0126, respectively, reduces ADM2-induced HTR-8SV/neo cell invasion and migration. In summary, this study suggests a potential involvement for ADM2 in regulating HLA-G antigen at the maternal-fetal interface in human pregnancy and facilitating trophoblast invasion and migration via MAPK3/1 phosphorylation.

Potential role of intermedin/adrenomedullin 2 in early embryonic development in rats

Adrenomedullin2 (ADM2), also referred to as Intermedin (IMD) is expressed in trophoblast cells in human placenta and enhances the invasion and migration of first trimester HTR-8/SV-neo cells. Recently we demonstrated that infusion of IMD antagonist in pregnant rats causes feto-placental growth restriction suggesting a role for IMD in maintaining a successful pregnancy. Therefore, this study was undertaken to assess if IMD has a functional role in embryo implantation in a rat model. We show that IMD mRNA is expressed in rat implantation sites and its expression is significantly higher on day 15 in placenta compared to days 18–22. Infusion of IMD antagonist IMD 17–47 from day 3 of pregnancy causes a significant decrease in the weights of day 9 implantation sites as well as serum levels of 17β-estradiol, progesterone, nitric oxide and serum MMP2 and MMP9 gelatinase activity. Further, expression of MMP2, MMP9, VEGF and PLGF protein levels are significantly downregulated in the implantation sites of IMD antagonist treated rats. This study suggests a potential involvement of IMD in regulating the factors that are critical for implantation and growth of the embryo and thus in establishment of normal rat pregnancy.

Characterization of the invasive and inflammatory traits of oral Campylobacter rectus in a murine model of fetoplacental growth restriction and in trophoblast cultures

Campylobacter species (C. jejuni, C. fetus) are enteric abortifacient bacteria in humans and ungulates. Campylobacter rectus is a periodontal pathogen associated with human fetal exposure and adverse pregnancy outcomes including preterm delivery. Experiments in pregnant mice have demonstrated that C. rectus can translocate from a distant site of infection to the placenta to induce fetal growth restriction and impair placental development. However, placental tissues from human, small-for-gestational age deliveries have not been reported to harbor C. rectus despite evidence of maternal infection and fetal exposure by fetal IgM response. This investigation examined the temporal relationship between the placental translocation of C. rectus and the effects on fetal growth in mice. BALB/c mice were infected at gestational day E7.5 to examine placental translocation of C. rectus by immunohistology. C. rectus significantly decreased fetoplacental weight at E14.5 and at E16.5. C. rectus was detected in 63% of placentas at E14.5, but not at E16.5. In in vitro trophoblast invasion assays, C. rectus was able to effectively invade human trophoblasts (BeWo) but not murine trophoblasts (SM9-1), and showed a trend for more invasiveness than C. jejuni. C. rectus challenge significantly upregulated both mRNA and protein levels of IL-6 and TNFalpha in a dose-dependent manner in human trophoblasts, but did not increase cytokine expression in murine cells, suggesting a correlation between invasion and cytokine activation. In conclusion, the trophoblast-invasive trait of C. rectus that appears limited to human trophoblasts may play a role in facilitating bacterial translocation and placental inflammation during early gestation.

Liver iron status and associated haematological parameters in relation to fetal growth and pregnancy outcome in rapidly growing adolescent sheep carrying a singleton lamb derived by embryo transfer

Maternal and fetal liver iron concentrations and associated haematology parameters were determined in an adolescent sheep paradigm characterised by rapid maternal growth, premature delivery and feto-placental growth restriction. Singleton-bearing dams were offered a control or high dietary intake to induce normal or growth-restricted pregnancies, respectively. Pregnancies were terminated on Day 90 or 130 of gestation or progressed to term. Relative blood volume increased (P < 0.05) and liver iron concentration decreased (P < 0.003) from mid to late gestation in control, but not in high-intake dams. At 90 and 130 days gestation, liver iron concentrations were reduced (P < 0.001) in high-intake dams but fetal liver iron was independent of dam nutrition. High intakes leading to poor pregnancy outcome at term were characterised by increased maternal haematocrit, haemoglobin, total plasma protein, albumin (all P < 0.001) and serum iron (P < 0.05), and by reduced oestradiol 17β (P < 0.001) at Day 130. Thus, high dietary intakes that promote rapid maternal growth and adiposity are associated with early depletion of maternal liver iron stores and a relative failure of normal blood volume expansion, which may, in turn, underlie the reduction in uteroplacental blood flows and fetal nutrient delivery previously established for this paradigm.

Characterization of the invasive and inflammatory traits of oral Campylobacter rectus in a murine model of fetoplacental growth restriction and in trophoblast cultures

Characterization of the invasive and inflammatory traits of oral Campylobacter rectus in a murine model of fetoplacental growth restriction and in trophoblast cultures

Expression of Adrenomedullin 2 (ADM2)/Intermedin (IMD) in Human Placenta: Role in Trophoblast Invasion and Migration1

Calcitonin gene-related peptide (CALCB), amylin, and adrenomedullin (ADM) belong to a unique group of calcitonin (CALCA)/CALCB family peptides that have overlapping biological effects owing to their structure and cross-reactivity between receptors. CALCB and ADM are expressed in fetoplacental tissues and are important in maintaining normal placental function. Recently, ADM 2 (ADM2)/intermedin was identified as a novel CALCA/CALCB family peptide that functions through CALCB and ADM receptors. ADM2 is expressed in the pituitary, digestive tract, and other organs of vertebrates and reduces blood pressure in both normal and hypertensive rats. We recently reported that the level of immunoreactive ADM2 is significantly upregulated in pregnant rats and that its hypotensive effects are also increased during rat pregnancy. Furthermore, infusion of ADM2 antagonist in pregnant rats causes fetoplacental growth restriction. The objective of this study was to analyze the expression and possible role of ADM2 in human placenta. We show that ADM2 mRNA is expressed in human placenta and that immunoreactive ADM2 is localized in syncytiotrophoblasts, cytotrophoblasts, and endothelial cells throughout human pregnancy. This study also demonstrates that ADM2 enhances the invasion and migration of first-trimester HTR-8SV/neo cells. ADM2 increases the invasive index of HTR-8SV/neo cells by 2.2-fold compared with controls. Taken together, the findings from this study suggest that ADM2 may have a role in the physiology of human pregnancy via regulation of trophoblast invasion and migration.

Adrenomedullin 2 Antagonist Infusion to Rats During Midgestation Causes Fetoplacental Growth Restriction Through Apoptosis1

Adrenomedullin 2 (ADM2) is a recently discovered member of the calcitonin/calcitonin gene-related peptide family with an exon-intron structure similar to that of ADM. The mRNA of ADM2 is expressed in several tissues, including uterus and ovary. The present study was designed to assess the effects of ADM2 antagonist (ADM2(17-47)) infusion to pregnant rats on fetal and placental growth. On Day 15 of gestation, rats were implanted s.c. with osmotic minipumps delivering 50 and 200 mug per rat per day of ADM2(17-47) and were killed on Gestational Day 18. In ADM2(17-47)-treated rats, placental weights were significantly inhibited in a dose-related manner, with an 11% reduction in the group of rats receiving 200 microg/day, whereas the fetal weights were reduced by 17% without significant differences between the two doses. 2 In ADM2(17-47)-infused rats, increased apoptosis was demonstrated in the labyrinth and junctional zones of rat placenta by the TUNEL method compared with the control animals. Western blot analysis demonstrated that in ADM2(17-47)-treated rats Bcl-2, mitochondrial cytochrome c, and active caspase-9 and caspase-3 were significantly increased compared with the controls. No significant treatment-associated changes were observed in Bax, Bid, p53, and caspase-8 and caspase-10 proteins in the treated placentas. In addition, infusion of ADM2(17-47) caused a significant decline in the transcripts of nitric oxide synthase 3 (NOS3) and NOS2. These findings show that ADM2(17-47) infusion in rats during midpregnancy cause fetoplacental growth restriction through the activation of mitochondrial apoptotic pathways. This study demonstrates for the first time (to our knowledge) a potential role for ADM2 in placental functions during pregnancy.

Effects of Parathyroid Hormone Like Hormone (PTHLH) Antagonist, PTHLH7–34, on Fetoplacental Development and Growth During Midgestation in Rats1

Parathyroid hormone-like hormone (PTHLH) secretion has been reported in human amnion, chorion, decidual cytotrophoblast, syncytiotrophoblast, endometrium, and myometrium; however, the functions of PTHLH during pregnancy, particularly during placenta formation and fetal development, are not well understood. We examined whether neutralization of PTHLH action using PTHLH antagonist, PTHLH(7-34), in rats during early gestation affects fetal and placental growth. Rats received s.c. a daily dose of either 0, 4, 12, or 36 microg of PTHLH(7-34) infused continuously through mini-osmotic pumps from Day 8 through Day 15 of pregnancy. Fetal weights measured on Day 15 were significantly decreased in rats treated with all the doses of PTHLH(7-34) compared to controls, and decreases in placental weights were significant at the 12-microg dose. TUNEL assay demonstrated an increased number of apoptotic cells in placenta of treated rats, including rats treated with the 4-microg dose. Cleaved caspase 3 (CASP3), caspase 9 (CASP9) (P < 0.05) and poly-ADP-ribose polymerase (PARP1) (P < 0.01) expression was increased and BCL2 (P < 0.01) expression was decreased in rats treated with 4 microg PTHLH(7-34) compared to that in control. Placental cytochrome c expression was increased (P < 0.01) in cytosolic and decreased (P < 0.01) in mitochondrial fraction in PTHLH(7-34)-treated rats. Caspase 8 expression was not affected by the treatment. Immunohistochemical analysis of platelet endothelial cell adhesion molecule (PECAM1) showed higher staining intensity in control than in treated rats. In conclusion, these results suggests that PTHLH plays a role in early pregnancy, and that antagonization of PTHLH action causes fetoplacental growth restriction through activation of mitochondrial pathway of apoptosis in the placenta and through decreased expression of PECAM1.

Role of pro-IGF-II processing by proprotein convertase 4 in human placental development

Fetal growth restriction (intrauterine growth restriction, IUGR) is a leading cause of perinatal mortality. However, the causes of aberrant development of the placenta and, thus, of the fetus, are not currently known. Insulin-like growth factor II (IGF-II) has been shown to be an important regulator of fetoplacental growth. This growth factor must undergo posttranslational processing, and, thus, we hypothesized that aberrant processing of pro-IGF-II to IGF-II may be a cause of IUGR. Here, we have found that the proprotein convertase PC4 is expressed in the human placenta and that it cleaves pro-IGF-II to generate the intermediate processed form, IGF-II (1-102) and, subsequently, mature IGF-II (1-67), which are accounted for by the removal of terminal basic residues by carboxypeptidases. This processing confers the ability of IGF-II to activate invasive trophoblast cells through AKT phosphorylation, whereas inhibition of PC4 by a PC4-specific inhibitor blocks pro-IGF-II processing and reduces trophoblast cell migration, which can be partly restored by addition of mature IGF-II. Consistent with the hypothesis that IGF-II processing is implicated in IUGR, sera of patients carrying IUGR fetuses displayed elevated levels of pro-IGF-II. Thus, abnormal processing of IGF-II by PC4 may represent a previously uncharacterized mechanism involved in the pathophysiology of fetoplacental growth restriction, and elevated pro-IGF-II may be a useful clinical marker for risk of IUGR.

Adrenomedullin antagonist treatment during early gestation in rats causes fetoplacental growth restriction through apoptosis.

Adrenomedullin (AM), a potent vasorelaxant peptide, has been shown to function as an angiogenic and growth factor. The present study investigated whether antagonism of endogenous AM in rats during early gestation results in diminished placental and fetal growth and whether this occurs through induction of apoptosis. Rats on Gestational Day 8 were implanted s.c. with osmotic minipumps delivering 125 and 250 microg rat(-1) day(-1) of AM(22-52) and were killed on Gestational Day 15. In AM(22-52)-treated rats, both placental and fetal weights were dose-dependently inhibited, with 50% reduction in the group receiving 250 microg rat(-1) day(-1). In these animals, fetal resorption sites were also increased. Apoptosis was demonstrated in placenta and uterus by the TUNEL method. Apoptotic changes were more apparent in trophoblast cells in the labyrinth zone of placenta and uterine decidua of AM(22-52)-treated rats when compared with vehicle-control rats. Immunoreactivity to active caspase-3 protein was abundant in the placenta and uterus of the AM(22-52)-treated group. Western blot analysis demonstrated that in homogenates of both the placenta and uterus of AM(22-52)-treated rats, levels of active caspase-9 and -3 as well as of Poly ADP ribose polymerase were significantly increased, whereas levels of Bcl-2 protein decreased, compared with controls. However, no significant treatment-associated changes were observed in Bid, Fas, Fas ligand, p53, and caspase-8 and -10 proteins in either placenta or uterus. Bad protein was undetectable in either tissue. In mitochondrial fractions from both placenta and uterus, the levels of Bax increased with decreases in cytochrome c on AM(22-52) treatment. Conversely, in the cytosol, Bax levels decreased with increases in cytochrome c, demonstrating translocation of Bax from cytosol to mitochondria and release of cytochrome c from mitochondria with AM(22-52) treatment. In conclusion, these findings show that antagonism of AM in rats during early pregnancy caused fetoplacental growth restriction through the activation of mitochondrial apoptotic pathways.

Bestatin results in pathophysiological changes similar to preeclampsia in rats via induction of placental apoptosis.

To establish a rat preeclampsia model with fetoplacental growth restriction caused by bestatin via induction of placental apoptosis. 200 mg/kg/day of bestatin or saline as a control were infused intraperitoneally into pregnant Wistar rats from 15 days' gestation. In the first experiment, maternal blood pressure and proteinuria were examined during the pre- and postpartum periods. In the second experiment, cesarean sections were performed at 20 days' gestation and the weights of pups and placentas, and levels of proteinuria and placental apoptosis were examined. Physiological decrease of blood pressure in late pregnancy was not detected in the bestatin group but proteinuria level at 20 days' gestation was elevated. The weights of pups and placentas in the bestatin group were significantly lower than those in the controls, bestatin strongly inducing apoptosis in the placenta. Bestatin may cause a preeclampsia-like condition through induction of placental apoptosis.