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.

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.

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.

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.

The missing link in amniotic fluid volume regulation: intramembranous absorption.

Although fetal urine output and swallowing are major contributors to amniotic fluid regulation, other pathways for fluid movement must be involved in the regulation of amniotic fluid volume because many studies report fetal urine output to be greater than swallowing. This study was designed to examine the possibility of fluid transfer between the amniotic cavity and the fetal blood that perfuses the fetal membranes and surface of the placenta in the ovine fetus. We injected warmed distilled water into the amniotic fluid in three groups of chronically catheterized fetal sheep. In normal fetuses, there was rapid absorption of the water into the fetal circulation, resulting in highly significant decreases in fetal osmolality, plasma electrolytes, and heart rate as well as increases in arterial pressure and fetal hemolysis. Concomitantly, there was a small, delayed fall in maternal osmolality. In a second group of fetuses with ligated esophagi, these same responses occurred except that the fetal osmolality and electrolyte changes occurred earlier and were significantly greater. In a third group of fetuses killed just before the water injection, maternal osmolality was unchanged. These data suggest the intramembranous pathway as a major route of amniotic fluid absorption in the ovine fetus. In addition, esophageal ligation appears to augment the conductance of this pathway, as evidenced by a significantly greater estimated filtration coefficient and rate of water absorption in the ligated animals than in controls. Finally, the transmembranous pathway directly to the mother does not appear to be a major route.

Amniotic fluid volume and its relationship to fetal fluid balance: review of experimental data.

The studies described above collectively suggest that, whenever there is a decrease in fluid balance in the fetus, both fetal urine flow and tracheal secretion into the amniotic space are decreased. Conversely, when fetal hydration is increased, both urine and tracheal flows into the amniotic fluid may be increased. These observations suggest the hypothesis that, except under pathological conditions, aberrations in amniotic fluid volume may be the consequence of the existing state of hydration of the fetus. In addition, it appears that under many circumstances, these deviations from normal in fetal fluid balance may be due to maternal influences. Of course, the concept that fetal fluid balance is the primary factor determining amniotic fluid volume needs to be modified to incorporate transmembrane fluxes. However, these fluxes have yet to be documented as they relate to amniotic fluid volume and its regulation.

Amniotic fluid volume and normal flows to and from the amniotic cavity.

The regulation of amniotic fluid volume and composition remains largely unknown. Several difficult to study pathways exist for the formation (early transfer across fetal skin, fetal urine and lung fluid production) and removal (swallowing, transmembranous and intramembranous absorption) of amniotic fluid. It appears that flows across these pathways may work in concert the vast majority of the time because amniotic fluid volume is maintained within a normal range. When significant deviations occur from this normal range, especially in the mid trimester, significant perinatal morbidity and mortality incidence results.

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.

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.

Fetal electrolyte and acid-base responses to amnioinfusion: lactated Ringer's versus normal saline in the ovine fetus.

OBJECTIVE: We hypothesized that amnioinfusion with normal saline would increase fetal plasma sodium and chloride concentrations, resulting in a hyperchloremic acidosis, and that these alterations would not occur after amnioinfusion with lactated Ringer's solution. METHODS: Chronically catheterized fetal sheep (137 +/- 1 days' gestation; mean +/- SE) were divided into three groups: control (n = 8), infused with normal saline (n = 10), and infused with lactated Ringer's solution (n = 10). The protocol consisted of a 30-minute pre-infusion period, a 1-hour amnioinfusion, and a 1-hour recovery period. During amnioinfusion, warmed solution was infused at a rate of 100 mL/minute for 1 hour. Fetal plasma and amniotic fluid electrolyte concentrations and osmolalities were measured every 20 minutes. Statistical analysis was by analysis of variance and linear regression. RESULTS: Amniotic fluid electrolyte concentrations changed significantly (P < .001) in both amnioinfusion groups, resulting in amniotic fluid compositions that were essentially the same as the infused fluid 20 minutes after starting the amnioinfusion. Significant increases in fetal plasma Na+ and CI- concentrations (2-3 mEq/L) occurred in the normal-saline infusion group relative to both the control and lactated Ringer's groups (P < .001). The lactated Ringer's group demonstrated only a modest increase in plasma Na+ (P = .04) and no change in plasma Cl- concentration. Fetal arterial pH decreased (-0.015 U) in the normal-saline group, and the change in fetal pH was linearly related to the change in plasma Cl- concentration (r = -0.532, P = .004). CONCLUSIONS: Normal-saline amnioinfusion can significantly alter fetal plasma electrolyte concentrations and blood pH, whereas amnioinfusion with lactated Ringer's solution results in minimal changes in fetal electrolytes and acid-base balance. The fetal plasma changes that occur during saline infusion are in the physiologic but not the pathologic range.

Changes in blood flow to the ovine chorion and amnion across gestation.

OBJECTIVE: The purpose of this study was to determine the developmental changes in blood flow to the network of fetal microvessels in ovine chorion and amnion. METHODS: Colored microspheres (15.10 +/- 0.02 [standard deviation] mu in diameter) were infused into the superior vena cava in nine chronically catheterized fetal sheep with gestational ages ranging from 103-141 days (term 147). After euthanasia, chorion, amnion, and cotyledons were separated and microspheres were counted to determine blood flow rates. Standard correlation and regression analyses were used to analyze the data. RESULTS: Chorionic blood flow rate increased linearly (r = 0.82, P = .006) from 12 mL/minute at 103 days' gestation to 70 mL/minute at 141 days, and averaged 10.3 +/- 1.2% of the total umbilical blood flow. Weight-normalized chorionic flow (18.4 +/- 2.0 [standard error] mL/minute/kg of fetus) did not change significantly across gestation. Absolute and weight-normalized blood flow to the amnion (0.82 +/- 0.31 mL/minute and 0.34 +/- 0.11 mL/minute/kg fetus) increased with advancing gestation until 130 days and declined thereafter. Absolute cotyledonary blood flow rate increased with gestational age (r = 0.81, P = .008), and weight-normalized cotyledonary blood flow decreased with advancing gestation (r = -0.89, P = .002). Absolute but not weight-normalized chorionic and cotyledonary blood flow rates correlated positively. CONCLUSIONS: Blood flow to the microvascular network in the ovine chorion is high and increases with advancing gestation. Blood flow to the amnion is low but not insignificant. Therefore, intramembranous exchange may play an increasingly important role in determining amniotic fluid volume and composition as gestation proceeds.

Arginine vasopressin-induced changes in blood flow to the ovine chorion, amnion, and placenta across gestation.

OBJECTIVES: To determine whether physiological increases in fetal plasma arginine vasopressin (AVP) concentration alter blood flow rates to the ovine chorion, amnion or placenta, and to determine whether these AVP-induced changes in flow are dependent on gestational age. METHODS: Colored microspheres (15.10 +/- 0.02 microns (standard deviation) were infused into the superior vena cava before and at 30, 75, and 125 minutes of an intravenous AVP infusion (3 ng/min/kg) in 9 chronically catheterized fetal sheep between 103 days to 141 days gestation (term = 147 days). Chorion, amnion, and placental cotyledons were removed, and microspheres were counted to determine blood flow rates. RESULTS: Fetal arterial pressure (FAP) increased (analysis of variance, P < .0001) and heart rate (FHR) decreased (P < .0001) during the infusion, with responses greater in older (> 125 days) compared to younger (< 125 days) fetuses (P < .001). Chorionic blood flow rate increased by 19 +/- 7% at 30 min of the AVP infusion, and declined by 18 +/- 6% at 75 min, and 32 +/- 5% at 125 min (P < .0001). Similarly, fetal placental blood flow rate increased by 20 +/- 7% at 30 min of infusion, and declined, in parallel with chorionic blood flow rates, by 6 +/- 4% at 75 min, and 17 +/- 4% at 125 min (P < .0001). Amniotic blood flow rate did not change significantly during the infusion. The membranous and placental blood flow rate responses to AVP infusion did not depend upon gestational age. CONCLUSIONS: Physiologic increases in plasma AVP concentration induce an early increase in chorionic blood flow followed by a gradual decrease which parallels similar changes in placental blood flow rate. Unlike FAP and FHR, these blood flow changes are not gestational age-dependent. AVP-induced blood flow changes could play an important role in determining abnormalities of amniotic fluid volume observed clinically in some stressed fetuses.

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%.

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.

Fetal blood volume responses to acute fetal hypoxia.

The purpose of this study was to explore the effects of acute hypoxia on the volume of blood circulating in the fetus. The oxygen content of air inspired by chronically catheterized, near-term pregnant sheep was reduced and supplemented with carbon dioxide in an attempt to maintain fetal carbon dioxide tension constant. Control fetal arterial oxygen tension averaged 22.3 +/- 0.7 (SE) mm Hg. During a 30-minute hypoxic period, reductions in fetal arterial oxygen tension by 2.4 to 12.5 mm Hg in individual animals were associated with decreases in fetoplacental blood volume of 1.1% to 14.3%. On the average (n = 14), fetal blood volume decreased by 6.7% +/- 0.8% when oxygen tension decreased by 7.7 +/- 0.9 mm Hg. Both arterial and venous pressures increased significantly during the hypoxic period. Upon the return to breathing of room air, there was a transient overshoot of fetal PO2 of 1.0 +/- 0.4 mm Hg at 10 minutes, but fetal blood volume did not return to normal until 30 minutes after the hypoxia. Thus, the data suggest that acute fetal hypoxia causes rapid and sustained reductions in fetal blood volume whereas volume returns to normal only slowly after elimination of the hypoxia.

Thoracic duct lymph flow and its measurement in chronically catheterized sheep fetus.

A method was developed for chronic catheterization of the left thoracic lymph duct at the base of the neck in the sheep fetus, which did not disrupt the other major lymphatic vessels that join the venous circulation at the same location. The lymphatic catheter was connected to a catheter in a jugular vein when lymph flow was not being recorded, so that the lymph continuously returned to the fetal circulation. Special consideration of catheter size to minimize flow resistance and treatment to prevent clotting were required. Individual animals were maintained up to 17 days with lymph flow continuing. In 13 fetuses averaging 128 days gestation (term = 147 days) at the time of catheterization, lymph flow rate was measured for 1 h each day for the first 7 postsurgical days with an on-line computer technique that continuously calculated lymph flow rate. Lymph flow averaged 0.64 +/- 0.17 (SD) ml/min in fetuses weighing 2.3-4 kg and tended to undergo a nonsignificant increase with time. Lymph and plasma protein concentrations did not change with time. In individual fetuses, large spontaneous variations in lymph flow rate occurred over periods of several seconds to a few minutes. Analysis showed that these variations in flow rate were not associated with fetal breathing movements. Thus the present study describes a technique for studying the dynamics of lymph flow in the unanesthetized sheep fetus in utero over a time period limited primarily by the length of gestation. In addition, it appears that thoracic duct lymph flow rate in the fetus per unit body weight averages three to four times that in the adult.

Effects of outflow pressure on fetal lymph flow.

The purpose of this study was to determine to what extent fetal thoracic duct lymph flow may be reduced by increases in fetal venous pressure. In pregnant sheep the fetal left thoracic lymph duct was catheterized at the base of the neck and this catheter was connected to a jugular-vein catheter so the lymph could spontaneously return to the fetal circulation. At 5 days after catheter implantation in nine unanesthetized fetuses at 133 +/- 1 (SE) days' gestation, lymph flow was measured by disconnecting the lymphatic catheter from that in the jugular vein and varying outflow pressure of the lymphatic catheter independent of venous pressure. Whenever outflow pressure was negative, lymph flow was independent of outflow pressure and averaged 0.66 +/- 0.05 ml/min. When outflow pressure of the left thoracic duct was increased above zero, lymph flow decreased linearly with outflow pressure and flow stopped at an outflow pressure of 11.5 +/- 0.6 mm Hg. At a normal venous pressure of 3 mm Hg, the lymph-flow sensitivity to venous pressure was such that a 1 mm Hg rise in venous pressure reduced lymph flow by 12.7% +/- 1.2%. Thus it appears that fetal lymph flow is very sensitive to outflow pressure and only moderate elevations in venous pressure potentially may lead to fetal edema.

Fetal blood volume responses to acute fetal hemorrhage.

The purpose of this study was to explore the changes in fetal blood volume that occur after fetal hemorrhage. Fifteen unanesthetized, chronically catheterized fetal sheep averaging 130 +/- 3 (SD) days gestation were studied 4 to 6 (average 5) days after catheter implantation. The fetuses were hemorrhaged by continuous withdrawal from an arterial catheter over 5 minutes. On the average (n = 15), 13.9 +/- 5.3% of the initial volume was removed. Fluid gradually entered the fetal circulation during and after the hemorrhage. Thirty minutes after hemorrhage had been initiated, 53.0 +/- 22.0% restitution of the lost volume occurred. Thus, short-term fetal blood volume restitution after fetal hemorrhage averaged about twice that of the adult. Hemorrhages averaging 7.3 +/- 2.3% (n = 3), 12.9 +/- 1.0% (n = 8), and 21.0 +/- 3.3% (n = 4) were followed by 55.7 +/- 13.3%, 54.7 +/- 26.4%, and 47.7 +/- 19.9% restitution, respectively. Thus, fractional volume replacement appears independent of the shed volume over the range of 5-25% volume loss. The protein concentration in the fluid which entered the fetal vasculature averaged 53% of the plasma protein concentration, suggesting that the fetal interstitium was the primary source of the fluid. In summary, the data suggest that the fetus is able to replace rapidly about half of the volume lost due to rapid hemorrhage, and appears considerably better at controlling its blood volume immediately after rapid hemorrhage than the adult.