Fetal Growth Restriction Induced by Transient Uterine Ischemia-Reperfusion: Differential Responses in Different Mouse Strains.

We characterized fetal and placental growth and uterine and placental inflammation in pregnant C3H/HeOuJ and C57BL/6J mice (strains with different sensitivities to metabolic and circulatory pathologies), using different uterine ischemia/reperfusion (I/R) protocols, to establish and refine a murine model of I/R-induced fetal growth restriction (FGR). Pregnant C3H/HeOuJ mice on gestation day 15 were subjected to unilateral uterine I/R by (1) total blood flow restriction (TFR) by occlusion of the right ovarian and uterine arteries for 30 minutes, (2) partial flow restriction (PFR) by occlusion of only the right ovarian artery for 30 minutes, or (3) sham surgery. Pregnant C57BL/6J mice were treated the same, but on gestation day 14 and with TFR for only 5 minutes due to high sensitivity of C57BL/6J mice to I/R. Four days post-I/R, the animals were euthanized to determine fetal and placental weight and fetal loss and to assay placental myeloperoxidase (MPO) activity. In C3H/HeOuJ mice, TFR/30 minutes induced significantly ( P < .05) lower fetal and placental weights and higher placental MPO activity, compared to controls. The PFR/30 minutes produced the same effects except placental weights were not reduced. In contrast, in C57BL/6J mice, TFR for only 5 minutes was sufficient to induce FGR and increase fetal loss; while PFR/30 minutes lowered fetal but not placental weights and increased fetal loss but not placental MPO activity. In summary, we present the first published model of I/R-induced FGR in mice. We find that mice of different strains have differing sensitivities to uterine I/R, therefore differing I/R response mechanisms.

Endothelin Receptor A Antagonism Prevents Damage to Glycogen-Rich Placental Cells Following Uterine Ischemia-Reperfusion in the Rat.

Fetal growth restriction (FGR) is a common cause of perinatal morbidity and mortality. Suboptimal uteroplacental perfusion is the most commonly identified cause of FGR, and ischemic lesions are often observed in placentas from pregnancies complicated by FGR. Ischemia followed by reperfusion is a strong stimulus to the production of the vasoconstrictor endothelin 1 (ET-1) which has been implicated in several models of FGR. We sought to investigate oxidative stress and placental morphology in a rat model of ischemia-reperfusion (I/R)-induced FGR and to evaluate the role of ET-1 in the observed pathology. Unilateral uterine I/R (30 min) was conducted, with and without simultaneous ET-1 receptor A (ETA) antagonism, on pregnant rats at gestation day 17. Placental tissues collected 24 hours later were evaluated immunohistochemically for oxidative damage. Tissue pathology was studied using quantitative morphometry. Glycogen-rich cellular areas in the placental junctional zone exhibited only 50% intact cells (P < .001) in both uterine horns following unilateral I/R, compared to controls. ETA antagonism prevented damage to the glycogen-rich cellular areas. Oxidative damage in response to I/R was prominent in the labyrinthine layer in both uterine horns and was not affected by ETA antagonism. We conclude that glycogen-rich cellular areas of the placental junctional zone are particularly vulnerable to damage from uterine I/R in the rat. Nucleic acid oxidative damage in the labyrinth is a prominent effect of uterine I/R. ETA antagonism protects placental cellular integrity during I/R challenge but does not prevent nucleic acid oxidative damage.

Endothelin Receptor A Antagonism and Fetal Growth in Endothelial Nitric Oxide Synthase Gene Knockout Maternal and Fetal Mice.

Fetal growth restriction (FGR) is commonly associated with perinatal morbidity and mortality. Nitric oxide (NO) deficiency increases endothelin-1 (ET-1) production, and this increased ET-1 may contribute to the pathophysiology of NO deficiency-induced FGR. Using an endothelial NO synthase knockout mouse model of FGR, we sought to determine (a) the relative importance of maternal versus conceptus (fetal and placental) NO deficiency and (b) the contribution of ET-1 to the pathogenesis of FGR in this model. Fetal growth restriction occurred both with NO-deficient conceptuses in the setting of maternal NO production and with maternal NO deficiency in the setting of NO-producing conceptuses. Placental ET-1 expression was increased in NO-deficient dams, ET receptor A (ETA) production increased in endothelial nitric oxide synthase(+/-) placentas, and antagonism of ETA prevented FGR. These results demonstrate that both maternal and conceptus NO deficiency can contribute to FGR and suggest a role for ETA antagonists as therapeutic agents in FGR.

Impact of toll-like receptor 4 deficiency on the response to uterine ischemia/reperfusion in mice.

Our objective was to determine the role of toll-like receptor 4 (TLR4) in uterine ischemia/reperfusion (I/R)-induced fetal growth restriction (FGR). Pregnant TLR4-deficient and wild-type mice were subjected to I/R or a sham procedure. Fetal and placental weights were recorded and tissues were collected. Pep-1 (inhibits low-molecular-weight hyaluronan (LMW-HA) binding to TLR4) was used to determine whether LMW-HA-TLR4 interaction has a role in FGR. TLR4-deficient mice exhibited significantly lower baseline fetal weights compared with wild-type mice (P<0.05), along with extensive placental calcification that was not present in wild-type mice. Following I/R, fetal and placental weights were significantly reduced in wild-type (P<0.05) but not in TLR4-deficient mice. However, I/R increased fetal loss (P<0.05) only in TLR4-deficient mice. Corresponding with the reduced fetal weights, uterine myeloperoxidase activity increased in wild-type mice (P<0.001), indicating an inflammatory response, which was absent in TLR4-deficient mice. TLR4 was shown to have a regulatory role for two anti-inflammatory cytokines: interferon-B1 decreased only in wild-type mice (P<0.01) and interleukin-10 increased only in TLR4-deficient mice (P<0.001), in response to I/R. Pep-1 completely prevented I/R-induced FGR (P<0.001), indicating a potential role for the endogenous TLR4 ligand LMW-HA in I/R-induced FGR. In conclusion, uterine I/R in pregnancy produces FGR that is dependent on TLR4 and endogenous ligand(s), including breakdown products of HA. In addition, TLR4 may play a role in preventing pregnancy loss after uterine I/R.

Unilateral uterine ischemia/reperfusion-induced bilateral fetal loss and fetal growth restriction in a murine model require intact complement component 5.

The role of complement in ischemia/reperfusion-induced fetal growth restriction and fetal loss is unknown. C5-deficient or wild type timed-pregnant mice were subjected to unilateral uterine ischemia/reperfusion on gestation day 13, either by (1) partial flow restriction by right ovarian artery clamping for 30 min, or (2) total flow restriction by clamping both ovarian and uterine arteries for 5 min. Ischemia/reperfusion-challenged pregnancy outcomes were compared to sham-operated controls 5 days later. Ischemia/reperfusion-treated wild type mice exhibited significantly increased bilateral fetal loss, which was greater in total flow restriction than in partial flow restriction, and decreased fetal weights, which were the same in total flow restriction and partial flow restriction for the surviving fetuses. Placental weights were unchanged by treatments. Ischemia/reperfusion increased uterine, but not placental, myeloperoxidase activity, which correlated with fetal loss. In contrast, C5-deficient mice were protected from both fetal growth restriction and fetal loss, and exhibited no increase in myeloperoxidase activity. These results demonstrate that unilateral uterine ischemia/reperfusion results in bilateral fetal loss and fetal growth restriction, mediated by a systemic mechanism. In the current model, this pathological process is completely dependent on intact complement component 5.

The significance of endothelin in platelet-activating factor-induced fetal growth restriction.

The significance of endothelin-1 (ET-1) in platelet-activating factor (PAF)-induced fetal growth restriction (FGR) was evaluated in timed-pregnant rats receiving intravenous carbamyl-PAF (c-PAF; 0.5, 1.0, or 2.5 µg/kg per h) or vehicle, with or without ET-1 receptor A (ET(A)) antagonist (10 or 20 mg/kg per d) for 7 days beginning on gestation day 14. Tissues were collected on day 21. Carbamyl-PAF reduced fetal weights dose dependently. Placental weights were significantly reduced but not dose dependently. ET(A) antagonism prevented FGR at the 0.5, but not the 1.0 and 2.5 µg/kg per h c-PAF doses. Correspondingly, placental, but not uterine, preproET-1 messenger RNA (mRNA) expression (determined by reverse transcription-polymerase chain reaction) was increased at 0.5 µg/kg per h but not at higher c-PAF doses. In summary, c-PAF infusion results in fetal and placental growth restriction in the rat. At low doses of c-PAF, ET-1 is central to the pathophysiology of PAF-induced FGR. At higher c-PAF doses, FGR is induced by mechanisms other than ET-1 action.

Endothelin receptor antagonist has limited access to the fetal compartment during chronic maternal administration late in pregnancy.

AIMS: Endothelin receptor A (ET(A)) antagonism normalizes fetal growth in several models of rodent fetal growth restriction (FGR). Our aims were to determine the levels of ET(A) antagonist in maternal and fetal plasma following chronic maternal administration, and to determine its impact on pregnancy outcome, survival and growth of rat pups. MAIN METHODS: Timed pregnant rats were treated with one of two endothelin receptor antagonists or vehicle, from gestation day 14-21 (term=22 days). The antagonists and their respective doses were ABT-546 (20mg/kg/day) and FR139317 (12 mg/kg/day). On day 21, in six rats per group, maternal and fetal plasma ABT-546 was assayed by HPLC. Five additional rats in each group delivered spontaneously and nursed their pups through postpartum day 7. Viability of newborns, oxygen saturation, litter sizes, and pup weights were recorded on postpartum days 1 and 7. KEY FINDINGS: Fetal antagonist levels reached only 2% of maternal levels (p<0.01). There were no significant differences among groups in length of gestation; litter size; survival, number and weight of live pups at birth and at 7 days postpartum; and tissue oxygen saturation. SIGNIFICANCE: Maternal administration of an ET(A) antagonist, at a dose sufficient to ameliorate FGR, has no adverse impact on survival and growth of neonatal rat pups. ET(A) antagonism, delivered maternally, produces sufficiently low fetal plasma levels of antagonist so as not to present a survival threat to the neonatal pups. The beneficial effects of maternally administered ET(A) antagonism on fetal growth occur in the maternal, not the fetal, compartment.

Pathophysiology of chronic nitric oxide synthase inhibition-induced fetal growth restriction in the rat.

OBJECTIVE: To evaluate the pathophysiology of chronic nitric oxide synthase (NOS) inhibition-induced fetal growth restriction (FGR) in the rat. METHODS: Timed-pregnant rats received L-NAME (2.5 mg/kg/h) with or without endothelin (ET-1) receptor A (ETA) antagonist from day 14 to 21 of gestation. In separate groups, ETA antagonist and/or L-NAME were discontinued on day 18. On day 21 fetal and placental weights, and maternal and fetal plasma nitrate/nitrite (NOx) were determined. RESULTS: L-NAME led to FGR, and decreased maternal and fetal NOx. Maternal NOx was further decreased when ETA antagonist was co-administered with L-NAME. ETA antagonism along with L-NAME did not impact fetal growth. Discontinuation of L­NAME on day 18 resulted in normal fetal and placental growth at day 21 and an increase of maternal NOx. Simultaneous cessation of both NOS inhibition and ETA antagonism on day 18 produced FGR at day 21, whereas continuation of ETA antagonism after discontinuation of L-NAME resulted in normal fetal growth. CONCLUSIONS: NOS inhibition in the pregnant rat leads to decreased maternal and fetal nitric oxide (NO) production and FGR. The effects of NOS inhibition on fetal growth are reversible, and are mediated at least in part by ET-1. With chronic NOS inhibition, ETA antagonism improves but does not normalize fetal growth, and may allow increased access of L-NAME to the fetal compartment. Continued access of L-NAME to the fetal compartment may limit the effect on fetal growth of any therapeutic intervention in this model of FGR.

Impact of endothelin A receptor antagonist selectivity in chronic nitric oxide synthase inhibition-induced fetal growth restriction in the rat.

OBJECTIVE: Endothelin receptor A (ETA) antagonism improves fetal and placental growth and placental perfusion on days 1 and 4, but not day 7 of a 7-day infusion of a nitric oxide synthase (NOS) inhibitor. Our purpose was to evaluate the significance of the degree of ETA antagonist selectivity on uteroplacental perfusion and fetal growth on day 7 of chronic NOS inhibition. METHODS: Timed-pregnant rats were treated with the NOS inhibitor nitro-L-arginine methyl ester (L-NAME, 2.5 mg/kg/h) with and without one of the following ETA antagonists or their respective vehicles for 7 days beginning on day 14 of gestation: A-127722 (2,000-fold selective for ETA over ETB), FR139317 (8,000-fold ETA-selective), or ABT-546 (28,000-fold ETA-selective). Uterine and placental perfusion, as well as fetal and placental weight, was evaluated at the 7th day of treatment (gestation day 21). RESULTS: L-NAME administration resulted in a significant reduction in uterine and placental perfusion as well as fetal and placental growth. In the setting of NOS inhibition, ETA antagonism did not improve uterine or placental perfusion or fetal growth after 7 days of infusion irrespective of the degree of selectivity of the antagonist used. CONCLUSIONS: ETA antagonism, irrespective of the degree of receptor selectivity, does not improve fetal growth or uteroplacental perfusion on day 7 of chronic NOS inhibition.

The fetal response to chronic placental insufficiency.

Fetal growth restriction is most commonly caused by failure of the placenta to meet the increasing demands for oxygen and substrate of the developing fetus, resulting in common fetal compensatory responses. Understanding these responses is helpful in developing a management strategy that will optimize pregnancy outcome.

Altered endothelin receptor binding in response to nitric oxide synthase inhibition in the pregnant rat.

The authors evaluate the expression of endothelin-1 (ET-1) and its receptors in the uterus and placenta during maternal nitric oxide synthase (NOS) inhibition. Timed-pregnant rats received L-NAME (2.5 mg/kg/h) or saline from day 14 to 21 of gestation. Uterine and placental tissues collected on day 21 were assayed for preproET-1, ET( A), and ET(B) mRNA expression; localization and expression of ET-1 and receptor proteins; and receptor activity. NOS inhibition did not affect preproET-1 mRNA expression in the placenta or uterus. ET(A) expression decreased in the uterine free wall, but no other changes in receptor mRNA expression were observed in the uterus or placenta. ET-1 and receptor proteins were unchanged. Placental ET(A) and ET(B) receptor binding decreased. Uterine ET(A) receptor binding decreased in the placental bed. ET-1, a prominent mediator during NOS inhibition, is not of uterine or placental origin. Reduced receptor binding activity is the primary means by which these tissues regulate their response to ET-1 in the setting of NOS inhibition.

Expression of endothelin 1 and its receptors in the hypoxic pregnant rat.

Endothelin 1 (EDN1) plays a primary role in the pathophysiology of hypoxia-induced fetal growth restriction in the rat. In this study we evaluated the effects of chronic maternal hypoxia on the expression of endothelin and its receptors and on receptor binding activity in the uterus and placenta of the rat, in order to elucidate their roles in hypoxia-induced fetal growth restriction. Timed-pregnant Sprague-Dawley rats were maintained in either a normoxic or a normobaric hypoxic (12% O(2)) atmosphere from Gestational Days 18-21. Uterine and placental tissues collected on Gestational Day 21 were assayed for Edn1, Ednra, and Ednrb (endothelin receptors) mRNA expression by real-time quantitative RT-PCR, for localization of EDN1 and its receptors by immunohistochemistry, for EDNRA and EDNRB protein expression by Western blot, and for receptor binding activity by homologous competitive binding assays. EDN1 mRNA expression was significantly increased in the hypoxic placenta, but not in the uterus, compared with normoxic controls. Immunohistochemistry revealed increased EDN1 specifically in the labyrinth of the placenta. Receptor mRNA levels were not significantly affected by hypoxia, but EDNRA protein expression was significantly decreased specifically in the uterine placental beds. Receptor binding decreased significantly in response to hypoxia in all tissues investigated, compared with controls. These results suggest that chronic maternal hypoxia results in increased expression of EDN1 in the placenta but not in the uterus, and that reduced binding activity, rather than regulation of receptor expression, is a mechanism by which these tissues regulate the local hemodynamic response to increased endogenous placental EDN1 in the setting of hypoxia.

Endothelin and platelet-activating factor: significance in the pathophysiology of ischemia/reperfusion-induced fetal growth restriction in the rat.

OBJECTIVE: The objective of the study was to evaluate the role of endothelin-1 and platelet-activating factor in ischemia/reperfusion-induced fetal growth restriction in the rat. STUDY DESIGN: On day 17 of gestation, the right uterine and ovarian arteries were occluded for 30 minutes in experimental but not sham-operated rats. All rats received endothelin receptor A antagonist, A-127722 (10 mg/kg per day), platelet-activating factor antagonist, WEB-2086 (1 mg/kg), or vehicle. On gestational day 21, litter size, fetal viability, and fetal and placental weights were recorded. Reverse transcription-polymerase chain reaction for phospholipase A2-IIA and preproendothelin-1 messenger ribonucleic acid was performed on uterus and placentas from each uterine horn. Groups were compared statistically by analysis of variance. RESULTS: Ischemia/reperfusion reduced fetal weights, in both the ischemic horn and the nonischemic horn (P < .001). Antagonism of either endothelin receptor A or platelet-activating factor normalized fetal growth in both horns. Neither placental weight nor the incidence of fetal demise was affected by ischemia/reperfusion. Phospholipase A2-IIA and preproendothelin-1 messenger ribonucleic acid expression did not differ between right and left uterine horns in any group. Uterine and placental tissues in the ischemia/reperfusion group exhibited increased phospholipase A2-IIA (P < .01) but not preproendothelin-1. CONCLUSION: Endothelin-1 and platelet-activating factor are both important mediators in the pathophysiology of ischemia/reperfusion-induced fetal growth restriction in the rat, contributing to the fetal growth restriction observed in both the ischemic and nonischemic horns. Antagonism of either mediator produces normal fetal growth in this model of fetal growth restriction.

Endothelin and the regulation of uterine and placental perfusion in hypoxia-induced fetal growth restriction.

OBJECTIVE: Normal placental function is dependent on maintenance of uteroplacental perfusion. Endothelin, a potent vasoconstrictor, is produced in and is active in the uteroplacental vasculature. The purpose of this study was to determine the role of endothelin in the regulation of uteroplacental perfusion under normal conditions, and in hypoxia-induced fetal growth restriction. METHODS: Timed-pregnant Sprague-Dawley rats, outfitted with arterial catheters, were maintained in either a normoxic or a normobaric hypoxic (12% oxygen) atmosphere from day 18 to 21 of gestation. During this time, the endothelin receptor A antagonist, A-127722, or its vehicle was administered. Regional blood flow was determined on gestational day 21 using 57Co-labeled microspheres. Data were analyzed by analysis of variance with statistical significance accepted at P<.05. RESULTS: Both placental and uterine placental bed perfusion were significantly decreased by hypoxia and returned to normal values with the endothelin antagonist (P<.01 and P<.05, respectively). Fetal weights were significantly lower in the hypoxic group (P<.001) and restored to control levels by the antagonist. CONCLUSION: In the rat, endothelin contributes little to the regulation of uteroplacental perfusion under normal conditions. Hypoxia results in a decrease in perfusion of the uteroplacental bed and of the placenta, and perfusion in both of these beds is normalized by endothelin A receptor antagonism. We conclude that endothelin plays a primary role in the pathophysiology of hypoxia-induced fetal growth restriction by reducing uteroplacental perfusion.

Infusion of exogenous platelet-activating factor produces intrauterine growth restriction in the rat.

OBJECTIVE: To determine the impact of exogenous platelet-activating factor (PAF) on pregnancy outcome in the rat. METHODS: Carbamyl-PAF (0.05, 0.5, or 5.0 microg/kg per hour) or vehicle was infused intravenously for 7 days by osmotic pump into timed pregnant rats. Infusion was begun on day 14 of a 22-day gestation. Maternal mean arterial blood pressures were measured on days 1, 4, and 7 of the infusion. On gestational day 21 (PAF infusion day 7), fetal and placental weights and viability were evaluated at hysterotomy. Uterine and placental PAF receptor expression was analyzed by reverse transcription-polymerase chain reaction and agarose gel electrophoresis. Data were analyzed by analysis of variance, chi(2), or the Mann-Whitney U test as appropriate. RESULTS: Fetal weights were dose-dependently lower than control, by 19% and 35%, respectively, at dosages of 0.5 and 5.0 microg/kg per hour (P < .001) but were not affected at the 0.05 microg/kg per hour dose of carbamyl-PAF. Placental weights were significantly lower at all doses (P < .001). Fetal demise was dose-dependently higher and was significantly different from the control group at the 0.5 and 5.0 microg/kg per hour doses of carbamyl-PAF (P < .0001). Maternal mean arterial pressures were not altered by these doses of carbamyl-PAF. PAF receptors were expressed abundantly in both uterus and placenta. CONCLUSIONS: Exogenous PAF produces dose-dependent fetal growth restriction in the rat. Placental growth is particularly sensitive to PAF and, coupled with the dose-dependent decline in fetal growth, suggests a dose-dependent decline in function. An elevated level of PAF is detrimental to fetal growth and well-being in the rat.

Prematurity in multiple gestations: identification of patients who are at low risk.

OBJECTIVE: The purpose of this study was to evaluate a strategy for the identification of patients with multiple gestations who are at low risk for preterm delivery. STUDY DESIGN: A prospective observational study among patients with twin and triplet gestations. At 20 and 24 weeks of gestation, screening for bacterial vaginosis and fetal fibronectin was performed, followed by digital and sonographic assessment of the cervix. The treating physicians were blinded to test results. RESULTS: At the 24-week examination, specificities for delivery at >32 weeks of gestation for digital examination (92.9%), fetal fibronectin level (93.9%), cervical length on sonographic scan (85.1%), and combined fetal fibronectin level and cervical length (81.3%) did not differ statistically. Negative predictive values for these tests were >or=95%. All tests performed better at 24 weeks of gestation than at 20 weeks of gestation. CONCLUSION: At 24 weeks of gestation, a normal digital examination, a negative fetal fibronectin level, a normal cervical length on sonographic scan, or the combination of a negative fetal fibronectin level and a normal cervical length each confer a similarly high likelihood of delivery at >32 weeks of gestation in women with multiple gestations.