Expression of erythropoietin mRNA, protein and receptor in ovine fetal membranes.

Erythropoietin and its receptor have been identified in human, murine and ovine placentas. Based on the common embryonic origin of the placenta and fetal membranes, we postulated that erythropoietin is similarly expressed in the fetal membranes. Using in situ hybridization and immunohistochemistry, we tested the hypothesis that ovine fetal membranes are sites of erythropoietin production and action. At 86, 103 and 138 days gestation, erythropoietin mRNA and protein were present in the amnion localized to the cell layer consisting largely of amniotic epithelium and in the chorion localized to the chorionic columnar cells consisting of cytotrophoblasts. Binucleate cells, differentiated cytotrophoblasts known to produce hormones, were identified in the chorion in the region of erythropoietin expression but were not observed in amniotic tissue. The erythropoietin receptor protein was present in the amnion and chorion at 103 and 138 days gestation but was not observed in either tissue at 86 days. In summary, erythropoietin appears to be produced as well as utilized within the ovine amnion and chorion. Within the amnion, the amniotic epithelial cells express the erythropoietin gene whereas, within the chorion, either the cytotrophoblasts or the binuclear cells may be the source. Due to the presence of the receptor, we speculate that the erythropoietin produced in the membranes may mediate fetal membrane function and/or growth through an autocrine and/or paracrine mechanism. Further, the fetal membranes may be the source of erythropoietin in the amniotic fluid.

Erythropoietin responses to progressive blood loss over 10 days in the ovine fetus.

Long-term loss of fetal blood can occur with fetomaternal hemorrhage, vasoprevia, or placental previa. Our objective was to determine the effects of progressive fetal blood loss over 10 days on fetal plasma erythropoietin (EPO) concentration and its relationship to arterial PO(2), hematocrit, and the volume of blood loss. Late-gestation fetal sheep (n = 8) were hemorrhaged daily at a rate of 1 ml/min over 10 days. The extent of hemorrhage differed in each fetus and ranged from 30 to 80 ml/day, with the cumulative volume removed ranging from 78 to 236 ml/kg estimated fetal weight. Four fetuses served as time controls. EPO concentration measurements were by radioimmunoassay. Statistical analyses included regression, correlation, and analysis of variance. We found that EPO and arterial PO(2) were unchanged until the cumulative hemorrhage volume exceeded 20-40 ml/kg. Once this threshold was exceeded, plasma EPO concentration increased progressively throughout the study and averaged 14.3 +/- 3.2 times basal values on day 10. EPO concentration, arterial PO(2), and hematocrit changes were related curvilinearly to cumulative hemorrhage volume (P < 0.01), whereas the relationship between plasma EPO and arterial PO(2) was log linear (P < 0.001). We conclude that 1) fetal plasma EPO concentration and arterial PO(2) are insensitive to a slow, mild-to-moderate blood loss over several days; 2) unlike the rapid return of EPO to normal within 48 h after acute hemorrhage, fetal EPO concentration undergoes a progressive increase with moderate-to-severe blood loss over several days; 3) the long-term hemorrhage-induced changes in EPO are best correlated with arterial PO(2); and 4) the fetal EPO response to hemorrhage does not appear to be limited by the fetus's ability to produce EPO.

Antibody-induced anemia in fetal sheep: model for hemolytic disease of the fetus and newborn.

OBJECTIVE: Our purpose was to produce a condition analogous to alloimmune hemolytic disease of the fetus and newborn by infusing antierythrocyte antibodies in fetal sheep. METHODS: Antierythrocyte antibodies were infused intravascularly into late-gestation ovine fetuses over a 10-day period. Fetal blood was sampled daily for complete blood cell counts, blood gases, iron, erythropoietin (EPO), and electrolyte concentrations. Red cell mass (RCM) and blood volume were determined every other day using indicator dilution techniques. Results were compared with eight similarly aged control animals. Statistical analysis included Student t test, three-factor analysis of variance, and least squares regression. RESULTS: The hematocrit in seven fetal sheep receiving antibody infusion declined significantly by 10.3 +/- 1.7%, whereas it increased in control animals 2.3 +/- 0.6% (P <.001). RCM was reduced by 18.9 +/- 3.2% over the 10-day protocol while increasing 34.1 +/- 4.2% in control animals, representing more than a 50% difference in RCM (P <.001). Fetal EPO was significantly increased with lower hematocrit and lower PO(2) (P <.001). As fetal hematocrit declined below 25%, lactate and reticulocytes also increased (P <.001). Plasma iron concentration was not significantly altered (P =.47). CONCLUSIONS: The chronically catheterized fetal sheep is a viable model for studying immunologically induced fetal anemia as hematocrit can be titrated and the fall in RCM and hematocrit are associated with fetal hypoxia and elevated EPO as occurs in the anemic human fetus. Furthermore, there appears to be a threshold degree of anemia required to elicit responses as the fetal EPO, PO(2), and lactate appeared unresponsive until hematocrit fell below 25%.

Erythrocyte and erythropoietin responses to hemorrhage in the immature and near term ovine fetus.

OBJECTIVE: Previous reports suggested that immature ovine fetuses have a greater erythropoietin response to hemorrhage than those near term. This study tested the hypothesis that immature ovine fetuses would expand their red cell mass more rapidly than near term fetuses after hemorrhage. STUDY DESIGN: Chronically catheterized immature ovine fetuses at 109.5 +/- 0.3 (mean +/- SE) days' gestation (term = 150 days) were studied over a 10-day period. They either underwent hemorrhage of 40% of their measured blood volume on day 3 or were in a time control group monitored without hemorrhage. Red cell mass, hematocrit, blood volume, plasma volume, and plasma erythropoietin concentrations were measured at 24- and 48-hour intervals. Responses in the immature fetuses were compared with responses in near term fetuses. RESULTS: In the control group red cell mass, hematocrit, blood, and plasma volumes increased significantly, whereas plasma erythropoietin concentration decreased significantly with advancing gestational age. In immature fetuses that underwent hemorrhage, the relative changes in red cell mass, hematocrit, and plasma erythropoietin concentration were not significantly different from those seen in the near term fetuses. The only significant posthemorrhage difference was that the increases in blood and plasma volumes were greater in the immature compared with the near term fetuses. CONCLUSION: Immature and mature fetuses have similar erythrocyte and erythropoietin responses to moderately severe hemorrhage. The larger blood and plasma volume responses in the immature fetuses are consistent with the concept that they have a greater extracellular fluid volume.

National Institute of Child Health and Development Conference summary: amniotic fluid biology--basic and clinical aspects.

This report summarizes the National Institute of Child Health and Development sponsored conference on amniotic fluid (AF) biology, held 28-29 September 1999, in Detroit, Michigan. National and international investigators with expertise in AF biology addressed the regulation of AF volume and composition as well as the clinical aspects of interpreting fetal health and well-being from AF indices. A major purpose of the meeting was to consider future directions and opportunities for basic and clinical research which focus on understanding the physiology and pathophysiology and providing therapeutic interventions for abnormalities of AF volume. To achieve this, the workshop participants addressed the current state of knowledge, recent scientific advances and priorities for major questions for which answers must be sought. The fact that it is not known whether AF volume is regulated or what volume-regulatory mechanisms might be involved is a major problem that needs addressing. In the later half of gestation, potential AF volume-regulatory pathways include the two major inflows into the amniotic compartment, i.e. fetal urine and lung liquid, and the two major outflows, i.e. fetal swallowing and intramembranous absorption. If AF volume is regulated, then this must occur through regulation of intramembranous flow, because the other three flows are regulated by the fetus to meet fetal needs. Regulation of AF composition is similarly unknown. In clinical practice, a variety of ultrasonographic indices of AF volume are used, but the relationships of these indices to AF volume have not been determined, nor have their dependency on fetal size, shape or position within the uterus. Further, although aberrations in AF volume both above and below normal are associated with increased fetal and neonatal morbidity and mortality, the predictive utility of the various AF indices remains low and there is little consensus on which is best utilized under conditions of oligohydramnios, normal AF volumes, or polyhydramnios. Further, various clinical AF therapies remain largely experimental and their optimization and utilization need exploration. This report is a condensation of the views presented by the conference participants.

Placental expression of erythropoietin mRNA, protein and receptor in sheep.

In ovine placentae at 100 days gestation, we localized the expression of erythropoietin (EPO) mRNA by in situ hybridization and determined the cellular localization of EPO protein and EPO receptor protein by fluorescence immunohistochemistry. Erythropoietin mRNA was observed in maternal tissue at the apical tips of the fetal cytotrophoblastic villi at their interface with the maternal caruncle and was absent from both the centrally located fetal-maternal tissue and the more peripherally located maternal caruncle. An EPO-protein-associated fluorescent signal was observed in the same interface region as the EPO mRNA hybridization signal. An intense fluorescent signal associated with EPO receptor protein was observed in the apical fetal-maternal interface region with a lower density signal dispersed throughout the remainder of the interdigitating fetal-maternal tissue. The predominant cells in the apical fetal-maternal interface were identified as binucleate cells by immunohistochemistry. Thus the localization of the binucleate cells was the same as that for the EPO mRNA and the EPO protein, whereas the EPO receptor had a more generalized distribution. Since the binucleate cells are hormone producing cells, we speculate that the binucleate cells are the source of the EPO that is present in ovine placenta.

Developmental expression of vascular endothelial growth factor (VEGF) receptors and VEGF binding in ovine placenta and fetal membranes.

The receptor tyrosine kinases, kinase-insert domain-containing receptor (KDR) and fms-like tyrosine kinase (Flt-1), and their ligand vascular endothelial growth factor (VEGF) are essential for the development and maintenance of placental vascular function during pregnancy. To further understand the role of VEGF in mediating angiogenesis and vascular permeability during development, the cellular localization of KDR and Flt-1 mRNA and protein, and the distribution of(125)I-VEGF binding sites in placenta, chorion and amnion of ovine fetuses were examined at three different gestational ages. In placentae at 62, 103 and 142 days, the predominant site of KDR mRNA and protein, and VEGF binding was the maternal vascular endothelium. In addition, a specific, although weak, signal for KDR mRNA was found in the maternal epithelium. At 103 and 142 days but not 62 days gestation, KDR mRNA and protein as well as VEGF binding sites were abundantly present in the endothelium of villous blood vessels. In the fetal membranes at 62, 103 and 142 days gestation, KDR mRNA and protein were expressed in the amniotic epithelium and intramembranous blood vessel endothelium, where binding of(125)I-VEGF was strong. There was no KDR mRNA or VEGF binding in the chorionic cytotrophoblast. Flt-1 expression was not detectable in placentae or fetal membranes at the three ages studied. In summary, the results demonstrated that VEGF receptors are present in the maternal and fetal vasculatures of the ovine placenta. This expression is consistent with a capillary growth-promoting function of KDR and its ligand VEGF. Further, the presence of KDR and VEGF binding sites in ovine fetal membranes suggests a role for VEGF in promoting intramembranous vascularity and permeability throughout gestation.

Increased urinary flow without development of polyhydramnios in response to prolonged hypoxia in the ovine fetus.

OBJECTIVE: In the ovine fetus subjected to 24 hours of hypoxia, urinary flow is normal within a few hours from the onset of hypoxia and there is a maintained inhibition of swallowing. We hypothesized that 4 days of fetal hypoxia would lead to polyhydramnios. STUDY DESIGN: Five late-gestation fetal sheep were subjected to hypoxia for 4 days and 7 other late-gestation fetal sheep served as time control animals. Fetal hypoxia was produced on postsurgical days 5 through 9 by continuous intratracheal nitrogen insufflation to the ewe. On days 3, 5, 7, and 9 after surgery, amniotic fluid volume, fetal urinary flow rate, and the compositions of maternal and fetal blood, amniotic fluid, and fetal urine were measured. A 3-factor analysis of variance was used for statistical analysis. RESULTS: During the period of experimental hypoxia the mean (+/-SE) fetal PaO(2) was 16.0 +/- 0.6 mm Hg, versus 21.2 +/- 0.7 mm Hg in control sheep (P <.001). Fetal hypoxia was associated with increased urinary flow on days 7 and 9, averaging 1410 +/- 310 and 2101 +/- 345 mL/d, respectively, versus 585 +/- 92 and 699 +/- 78 mL/d, respectively, in control animals (P <.001). Amniotic fluid volume was unchanged with time and averaged 960 +/- 159 mL in hypoxic fetuses on postsurgical days 7 through 9 and 851 +/- 130 mL in control animals (P =.60). Fetal blood lactate increased in the hypoxic animals, averaging 3.4 +/- 2.1 mmol/L versus 1.6 +/- 0.3 mmol/L in control animals (P =.02). Fetal urinary excretions of sodium, potassium, chloride, and lactate increased significantly during hypoxia, by 170% to 400%. CONCLUSION: Four days of nitrogen-induced hypoxia in the ovine fetus resulted in excess fetal urinary flow approximating 1000 mL/d greater than normal without the development of polyhydramnios. Because amniotic fluid volume did not change and hypoxia is a known inhibitor of fetal swallowing, we speculate that intramembranous absorption of amniotic water, electrolytes, and lactate increased.

Fetal esophageal ligation induces expression of vascular endothelial growth factor messenger ribonucleic acid in fetal membranes.

OBJECTIVE: Obstruction of the fetal esophagus does not always produce the expected polyhydramnios. This is because of increased intramembranous absorption of amniotic fluid into the fetal circulation. A possible mediator for this increased absorption is vascular endothelial growth factor (VEGF). The present objective was to explore whether VEGF gene expression and action would be induced in fetal membranes and placentas of ovine fetuses after esophageal ligation. STUDY DESIGN: Five late-gestation fetal sheep underwent esophageal ligation and 5 served as control animals. On postoperative day 9, amnion, chorion, and placenta were collected for cellular localization and quantitation of VEGF messenger ribonucleic acid by in situ hybridization and Northern blot analysis. Reverse-transcription polymerase chain reaction was used to identify the VEGF molecular forms. Immunostaining with Ki-67 antibody was used to determine the proliferation of vascular endothelium in the fetal membranes and placentas. RESULTS: VEGF messenger ribonucleic acid was localized in amniotic epithelium, chorionic cytotrophoblast, and cytotrophoblast of the placenta. VEGF164 was the major transcript expressed in these tissues. The abundance of VEGF messenger ribonucleic acid in the amnion and chorion, but not in the placenta, was significantly increased in the ligated fetuses in comparison with the control fetuses. The proliferation of the intramembranous blood vessel endothelium was greater in the ligated fetuses than in the control fetuses. CONCLUSION: The levels of VEGF messenger ribonucleic acid and the proliferation of vascular endothelium in the amnion and chorion increased after fetal esophageal ligation. This provides a possible mechanism for the enhanced intramembranous absorption of amniotic fluid through increased vascularity and permeability of the fetal membranes, thus ameliorating the development of polyhydramnios. We speculate that the signal(s) that mediate the increase in VEGF expression is present in either the fetal urine or the fetal lung secretions, or both.

Red blood cell life span in the ovine fetus.

Red cell life span within the fetal circulation has not been reported, although erythrocyte life span has been studied in the adult and newborn. The present study quantified red cell life span in 12 chronically catheterized fetal sheep at 97-136 days gestation (term = 150 days) with the use of autologous red cells labeled with [(14)C]cyanate. Cyanate forms a permanent covalent bond with hemoglobin and acts as a permanent red cell label. In the fetuses, blood (14)C activity decreased in a curvilinear fashion with time and reached 50% of the initial activity at 16.4 +/- 1.6 (SE) days. In contrast, (14)C activity of autologous red cells in two adult ewes decreased linearly with time as expected, reached 50% of the initial (14)C activity in 59 days, and yielded life spans of 117 and 121 days. Computer modeling and parameter optimization taking into account growth and skewed life span distribution were used to analyze the (14)C disappearance curve in each fetus. The mean life span of all red cells in the fetal circulation was 63.6 +/- 5.8 days. Mean red cell life span increased linearly from 35 to 107 days as fetal age increased from 97 to 136 days (r = 0.83, P < 0.001). Life span of cells produced at the time of labeling was significantly greater than the mean life span. Fetal growth rate estimated from parameter optimization was 3.28 +/- 0.72%/day; this compared well with the rate of 3.40 +/- 0.14%/day calculated from fetal weights at autopsy. Mean corpuscular volume decreased as a function of gestational age, but the decrease was small compared with the large increase in red cell life span. We conclude the following: 1) red cell life span in the fetal circulation is short compared with the adult; 2) red cells in younger fetuses have shorter life spans than in near-term fetuses; 3) the curvilinear disappearance of labeled red cells in the fetus appears to be due primarily to an expanding blood volume with fetal growth; and 4) red blood cell life span in a growing organism will be significantly underestimated unless the expansion of blood volume with growth is taken into account.

Amniotic fluid volume responses to intra-amniotic infusion of lactate in fetal sheep.

OBJECTIVE: In human and ovine fetuses, severe anemia is associated with elevated fetal blood and amniotic lactate levels and polyhydramnios. In ovine fetuses, intravascular infusion of sodium lactate elevates fetal plasma and amniotic lactate levels and produces polyhydramnios. The present study tested the hypothesis that an elevated amniotic lactate concentration in the absence of an increased fetal plasma lactate would be associated with an increase in amniotic fluid volume (AFV). METHODS: Eight chronically catheterized, late-gestation fetal sheep were studied over 5 days. Twice each day, we measured blood gases and pH, electrolytes, glucose, lactate, and blood urea nitrogen (BUN) concentrations, as well as osmolality of fetal blood, maternal blood, amniotic fluid, and fetal urine. Amniotic fluid volume was measured once daily. During experimental days 2 to 4, lactic acid was infused into the amniotic compartment to achieve an amniotic lactate concentration of approximately 20 mmol/L. Statistical analysis was by analysis of variance and regression. RESULTS: Amniotic fluid lactate levels averaged 2.2 +/- 0.4 mmol/L (mean +/- standard error) before infusion and 18.9 +/- 3.3 mmol/L during the 72-hour infusion, falling to 5.8 +/- 1.2 mmol/L postinfusion (P < .001). Fetal plasma lactate averaged 1.8 +/- 0.1 mmol/L on day 1 and increased by 1.4 +/- 0.6 mmol/L on day 4 (P < .001). Fetal urine flow was unchanged and averaged 0.54 +/- 0.08 mL/min over the 5 days. Amniotic fetal volume was 821 +/- 186 mL on day 1, increased nonsignificantly by 99 +/- 95 mL on day 4, and remained unchanged on day 5. CONCLUSIONS: The present study suggests that if amniotic lactate acts osmotically to increase AFV, the effect is small. Thus, the primary site of action of elevated fetal lactate levels appears to be at the placenta rather than the intramembranous pathway.

Effect of esophageal ligation on amniotic fluid volume and urinary flow rate in fetal sheep.

OBJECTIVE: Although the fetus normally swallows large volumes of amniotic fluid each day, it is unclear whether amniotic fluid volume increases after fetal esophageal obstruction or whether fetal urine production changes. Our objective was to determine the effects of fetal esophageal ligation on amniotic fluid volume and urinary flow rate over time. STUDY DESIGN: Seven late-gestation fetal sheep underwent esophageal ligation, and 7 served as time control animals. The urachus was ligated to eliminate urine flow to the allantoic cavity. On days 1, 3, 5, 7, and 9 after surgery, we measured the composition of amniotic fluid, fetal urine, and fetal and maternal blood, as well as amniotic fluid volume and fetal urinary flow rate. A 3-factor analysis of variance was used for statistical analysis. RESULTS: Amniotic fluid volume did not change with time in the control group, averaging 876 +/- 142 mL (mean +/- SEM), and it decreased in the esophageal ligation group (P =.020), averaging 309 +/- 75 mL on day 9. Fetal urinary flow rate was lower (P =.0063) in the esophageal ligation group (431 +/- 27 mL/d) than in the control group (631 +/- 54 mL/d). There were no differences in fetal or maternal blood compositions between the two groups. Amniotic fluid sodium and chloride increased in the ligated animals. CONCLUSION: Polyhydramnios did not occur after esophageal ligation, even though the fetuses excreted approximately 4000 mL of urine over the 9-day study period. This suggests that intramembranous absorption is substantially increased. With only small changes in amniotic solute concentrations, intramembranous solute absorption must occur simultaneously with water, suggesting a near-zero reflection coefficient for solutes. We speculate that fetal urine, lung secretions, or both contain a factor that increases intramembranous permeability.

Cellular localization of vascular endothelial growth factor in ovine placenta and fetal membranes.

To further understand the role of vascular endothelial growth factor (VEGF) in mediating angiogenesis and vascular permeability during development in the sheep placenta and fetal membranes, we examined the localization of VEGF mRNA and protein in placental, chorionic and amniotic tissues by in situ hybridization and immunohistochemistry in ovine fetuses at 62, 102 and 141 days gestation (term=150 days). In the placenta, VEGF mRNA expression and VEGF protein immunostaining were strong in cytotrophoblasts surrounding the villi. In addition, VEGF protein was localized in smooth muscle cells around fetal and maternal blood vessels and in the maternal epithelium. There was no apparent difference in placental VEGF mRNA or protein levels associated with advancing gestation. In the fetal membranes, VEGF mRNA was detected in the amniotic epithelium and the chorionic cytotrophoblastic cell layer. The intensity of the hybridization signals in both amnion and chorion appeared low at 62 days, moderate at 102 days and high at 141 days gestation. VEGF protein was detected in amniotic epithelium and chorionic cytotrophoblasts at all gestational ages studied. The increase in VEGF gene expression in fetal membranes as term approaches suggests that during fetal development VEGF may promote the vascularity and permeability of the microvessels which perfuse the fetal membranes, as well as permeability of the amniotic membrane itself. Thus VEGF may participate in the regulation of amniotic fluid volume.

Effect of laboratory acclimation on food and water consumption of pregnant sheep after fetal catheterization.

The objectives of this study were to determine 1) the time required for food and water consumption of late-gestation pregnant sheep to stabilize after a 6- to 7-h shipment by truck and 2) whether the duration of laboratory acclimation altered food and/or water consumption of pregnant sheep after fetal and maternal vascular catheterization. We used a semi-quantitative scale and a retrospective study design to determine food and water consumption as a function of acclimation time in post-shipping and post-surgery animals. These animals had been in our research facility for 2, 3, 4, and 5 or more days prior to surgical catheterization of the fetus and mother. We used a quantitative scale and a prospective study design to determine food and water consumption in post-surgery animals that had been in the laboratory for either 2 or > or = 7 days at the time of surgery. We used two- and three-factor repeated measures analyses of variance to determine the statistical significance of any differences. Although food and water consumption in post-shipping animals were significantly (p < 0.001) lower on day 1 than other days, we attributed this difference to the fact that "day 1" was shorter than 24 hours because the animals arrived in the laboratory at noon. Further, the post-surgery decrease and subsequent recovery in food and water consumption did not depend on the duration of the acclimation prior to surgery. We conclude that differences in pre-surgery, post-transportation acclimation periods ranging from 48 hours to > or = 7 days do not affect post-operative recovery from fetal surgery in sheep.

Relationship between graded degrees of anemia and amniotic fluid volume in the ovine fetus.

OBJECTIVE: Severe fetal anemia is associated with polyhydramnios in both human and ovine fetuses. This study examined the relationship between varying degrees of anemia and amniotic fluid volume in fetal sheep. STUDY DESIGN: Eleven long-term catheterized ovine fetuses at 126 +/- 1 days' gestation (mean +/- SE) were subjected to hemorrhage of 20 to 80 mL daily for 9 consecutive days to produce varying degrees of fetal anemia. Five additional animals served as time control animals. Statistical analysis was by least squares regression and 3-factor analysis of variance. RESULTS: Amniotic fluid volume was 793 +/- 147 mL and did not change with time in the control fetuses. In the fetuses that were subjected to hemorrhage the amniotic fluid volume changed little through the hematocrit range of 40% to 25%. As fetal hematocrit fell below approximately 25%, amniotic fluid volume began to increase. With greater degrees of anemia the amniotic fluid volume increased as an exponential function of hematocrit and approached 2000 mL excess fluid as hematocrit dropped to <15%. According to bivariate regression the increase in amniotic fluid volume was related to fetal hematocrit, PaO(2), and urinary flow rate as well as to plasma and amniotic fluid lactate concentrations. According to multivariate regression only fetal PO(2) and urinary flow rate were significantly related to the increase in amniotic fluid volume. CONCLUSIONS: Although mild anemia was not associated with increased amniotic fluid volume, moderate to severe fetal anemia was associated with an exponential rise in amniotic fluid volume. This rise may have been mediated by a hypoxemia-induced diuresis, by a diuresis related to a lactate-induced osmotic accumulation of fetal fluid, or by both mechanisms.

Developmental expression of vascular endothelial growth factor and its receptors in ovine placenta and fetal membranes.

OBJECTIVES: Vascularity of the surface of the placenta in humans and of the placenta and fetal membranes in several species including sheep is an important determinant of intramembranous absorption of amniotic fluid. Our previous studies have shown that the total blood vessel surface area in ovine amnion and chorion increases with advancing gestation. Vascular endothelial growth factor (VEGF) is a potent angiogenic and permeability factor and is found to be expressed in the ovine placenta and fetal membranes. To investigate the role of VEGF in maintaining the absorptive function of the intramembranous microvessels, the present study was undertaken to determine the gestational change in gene expression of VEGF and its receptors, kinase insert domain-containing receptor (KDR) and fms-like tyrosine kinase (Flt-1), in ovine placenta, chorion, and amnion. METHODS: Total RNA was extracted from placental cotyledon, chorion, and amnion of ovine fetuses at 60-140 days of gestation. The relative abundance of VEGF, KDR, and Flt-1 mRNA was determined by Northern blot analysis, and VEGF molecular forms expressed were identified by reverse transcriptase polymerase chain reaction. The gestational changes in mRNA levels of VEGF and its receptors were analyzed by regression analysis. RESULTS: In ovine placenta, chorion, and amnion, VEGF mRNA levels increased significantly from 60 to 140 days. The major VEGF molecular form expressed in these tissues was VEGF164, whereas VEGF120, VEGF144, and VEGF188 were present at lower levels. In the placenta, KDR was the primary VEGF receptor expressed, although Flt-1 was also detected at very low levels. In the amnion and chorion, KDR was the only receptor expressed. A gestational-dependent change in VEGF receptor expression was not observed in the placenta and membranes. CONCLUSIONS: The increase in VEGF gene expression with advancing gestation in the amnion and chorion where KDR is expressed suggests that VEGF and its receptor are important determinants of vascularity and permeability, and thus exchange capacity, of the intramembranous pathway.

Correction of hemorrhage-induced anemia with intra-amniotic iron in the ovine fetus.

OBJECTIVE: This study tested the hypothesis that intra-amniotic iron treatment would enhance fetal red blood cell production after an acute, severe fetal hemorrhage of 40% of estimated blood volume over 2 hours. STUDY DESIGN: Three groups of late-gestation ovine fetuses were studied for 10 days: (1) control fetuses (n = 8), (2) fetuses hemorrhaged on day 3 (n = 11), and (3) similarly hemorrhaged fetuses supplemented with a single bolus of 60 mg of iron injected intra-amniotically immediately after the hemorrhage (n = 7). Statistical analysis was by 3-factor analysis of variance. RESULTS: At 24 hours after hemorrhage, red blood cell mass increased 5% in the control group and was reduced equally in both hemorrhage groups by 32% below day 3 prehemorrhage values. At 7 days after hemorrhage, red blood cell mass increased 27.8% +/- 2.6% (SE) above day 3 baseline values in the control fetuses. In the nonsupplemented hemorrhaged fetuses, red blood cell mass was not different from prehemorrhage values after 7 days (+3.7% +/- 4.1%), whereas red blood cell mass increased by 29.9% +/- 6.1% above prehemorrhage values in the iron-supplemented hemorrhage group (P <.001). CONCLUSION: Intra-amniotic iron supplementation resulted in full restoration of red blood cell mass within 7 days after a large loss of blood in fetal sheep, whereas restoration failed without iron supplementation. Intra-amniotic iron treatment may be of therapeutic value in restoring red blood cell mass in human fetuses with certain types of anemia such as that resulting from fetal or fetomaternal hemorrhage.

Ovine vascular endothelial growth factor: nucleotide sequence and expression in fetal tissues.

To examine the role of vascular endothelial growth factor (VEGF) in mediating angiogenesis and vascular permeability during fetal development, we determined the gene expression of VEGF in ovine fetal tissues. Further, we cloned and sequenced the ovine VEGF cDNA encoding VEGF164 from sheep placenta. VEGF protein was localized in epithelial cells of the placenta and fetal kidney, and in hepatocytes of the fetal liver. By Northern analysis, a major VEGF mRNA species of 3.7 kb was identified in all tissues examined, with abundance highest in the lung and lowest in the liver. The most prominent molecular form expressed in ovine fetal tissues appeared to be VEGF164 with low levels of expression of VEGF120 and VEGF188. Cloning and sequence analysis of the most abundant form of ovine VEGF cDNA in the placenta confirmed the prediction of a 164-amino acid peptide, with a putative N-terminal signal sequence of 26 amino acids. Comparison of the VEGF cDNA sequence among different species revealed that VEGF is highly conserved suggesting an important role in development.

Fetal plasma iron and restoration of red blood cell mass after hemorrhage of the ovine fetus.

OBJECTIVE: Our purpose was to determine whether the restoration of fetal red blood cell mass after acute hemorrhage of 40% of the fetal blood volume is related to fetal plasma iron concentration. STUDY DESIGN: Ten chronically catheterized ovine fetuses were monitored for 10 days beginning at 125 +/- 1 (SE) days of gestation. After a 3-day control period 40% of the fetal blood was removed over 2 hours at a rate of approximately 1 ml/min. Fetal plasma iron and erythropoietin concentrations, hematocrit, blood volume, and red blood cell mass were measured daily before and for 7 days after fetal hemorrhage. Statistical analysis was by analysis of variance, correlation, and regression. RESULTS: Although blood volume was restored within 3 days of the hemorrhage (101.0% +/- 1.4% of prehemorrhage volume), red blood cell mass was not (81.8% +/- 2.8%). Only 6 of 10 fetuses restored their red blood cell mass to prehemorrhage levels by the end of the 7-day posthemorrhage period. On day 10 red blood cell mass correlated positively with prehemorrhage (r = 0.74, p = 0.015) and posthemorrhage (r = 0.69, p = 0.045) plasma iron concentration and negatively with posthemorrhage erythropoietin concentration (r = -0.68, p = 0.047). CONCLUSION: Fetal plasma iron concentration is an important factor in restoration of fetal red blood cell mass after loss of blood. The negative correlation of erythropoietin concentration with posthemorrhagic red blood cell mass suggests that iron, not erythropoietin, may be the limiting factor in recovery from hemorrhage-induced anemia. Thus iron supplementation of the fetus may be of benefit in the treatment of some types of fetal anemia.