INTRODUCTION

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IN THE SHEEP, the inflows of fluid into the amnion are fetal urination and fetal lung fluid production, and the outflows are fetal swallowing and fluid absorption into the vasculature of the amniochorion (4). Direct exchange between amniotic fluid and maternal plasma is quantitatively insignificant (1, 2, 4). Experimentally induced changes in amniotic fluid volume in sheep are not followed by appropriate changes in lung fluid production (7) nor in urine production (9). It follows that, at least under such experimental conditions, neither lung fluid production nor urination serves a regulatory role in the control of amniotic fluid volume. Whether fetal swallowing serves a regulatory role remains possible but inconclusive (15). There is evidence, however, that amniochorionic absorption in sheep increases appropriately when swallowing is not possible (13) and also when the amniotic fluid inflow is experimentally augmented by amniotic infusion of Ringer solution (9). It was proposed therefore that amniochorionic absorption is homeostatic in the service of volume control (9).

Although the site of amniochorionic absorption in sheep is believed to be the microvascular bed of the chorion (5), the question how this amniochorionic absorption is effected remains unanswered. Amniotic fluid absorption is often stated to be osmotic, because amniotic fluid is generally somewhat hypoosmotic with respect to fetal plasma, but direct evidence seems to be lacking. It is also possible that absorption depends on the difference in colloid osmotic pressures between amniotic fluid and fetal plasma. In the present experiments we investigated these possibilities by measuring amniochorionic absorption in the presence of induced changes in the osmolal and oncotic gradients between amniotic fluid and fetal plasma.

	METHODS

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Surgical preparation. The surgical and experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC). Ten time-bred ewes carrying singleton lambs were fasted for 24 h with access to drinking water and were operated on at 120 ± 1.5 (SD) days of gestation. Anesthesia was induced with an intravenous dose of 400 mg ketamine and 10 mg diazepam. The ewes were then intubated, and anesthesia was continued with a 50:50 mixture of oxygen and nitrous oxide and about 1% halothane. The concentration of halothane was adjusted to ensure a surgical level of anesthesia in both the ewe and the fetal lamb.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Schematic of the surgical preparation. See text for additional details. Indwelling intravascular catheters not shown. When fluids other than urine were infused, a second line in the roller pump was connected to the catheter leading into the amniotic fluid, and the urine was measured and sampled but not returned to the amnion.

Experimental protocols. The experiments began with a measured volume of amniotic fluid of known osmolality. The subsequent inflow of fluid into the amnion was monitored or controlled. The sum of the initial and inflow volumes constituted the volume of fluid to be accounted for at the end of each experiment. Thus the volume of fluid retrieved at the end of the experiment showed the volume of fluid that had been absorbed (or produced) by the amniochorion in the course of the experiment.

Instrumentation. Pressures were measured by means of Abbott Transpac-IV transducers and a Gould CL800232 polygraph or an InstruNet system, operated by GW Superscope II software installed in a MacIntosh G3 computer. Before each experiment, the transducers were calibrated against a mercury manometer. Vascular pressures were referred to amniotic fluid pressure. Arterial blood oxygen saturations, contents, pH, and gas pressures were measured in an Instrumentation Laboratories 842 CO-Oximeter and an Instrumentation Laboratories 1610 pH/Blood Gas Analyzer. Osmolalities were determined by freezing point depression in an Advanced model 3300 Micro Osmometer. Activities of ¹²⁵I were obtained with a Wallac 1470 Wizard gamma spectrometer.

Solutions. Solutions of various concentrations (see RESULTS AND DISCUSSION) of NaCl (Mallinckrodt) and mannitol (Sigma) were made with distilled water and sterilized in an autoclave. Plasma albumin solutions were prepared by dissolving lyophilized bovine serum albumin (Sigma A7906) into NaCl or mannitol solutions and were sterilized by filtration through sterile Corning no. 430770 0.45 micrometer cellulose acetate membranes.

Analytic methods. Total protein concentrations were determined by means of a commercial version of the Lowry method (Sigma Diagnostics). ¹²⁵I-labeled albumin concentrations were determined after the removal of small concentrations of loose label by precipitation in equal volumes of 10% trichloracetic acid.

Data presentation and statistical methods. Data are given as means ± SE, unless otherwise noted. Statistical calculations were performed by the GraphPad Prism version 3.02 for Windows, GraphPad Software (San Diego, CA) installed in a Dell dimension 4100PC. Tests used are specified in RESULTS AND DISCUSSION.

	RESULTS AND DISCUSSION

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Condition of the animals. Twenty-one experiments were performed on 10 fetuses. The average duration of the experiments was 66 ± 5 h (2.7 days). Table 1 shows the fetal control data before each of the 21 experiments were begun. The control data before a second or third experiment on the same fetus were compared with the control data before the first experiment by paired t-tests, and no statistically significant changes were found in any of the parameters listed in Table 1, indicating that the experiments did not affect the health of the fetuses. Mean autopsy weight of the fetuses was 3.84 ± 0.37 kg at a gestational age of 132 ± 2 days, which is normal, and no fetal abnormalities, other than those surgically created, were found at postmortem examination.

Crystalloid osmolality and amniochorionic absorption. In five control experiments, the drained amniotic fluid was reinfused, and the drained urine was infused also. The osmolality differences between amniotic fluid and fetal plasma in these experiments were recorded but not experimentally controlled. In these experiments, mean fluid absorption was 21.9 ± 3.6 ml/h. This is in the range of previously recorded flow rates, as summarized in Ref. 9, indicating that the minor surgical modifications of our original preparation had no effect on amniochorionic absorption. These data are shown as squares in Fig. 2.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Least squares linear regression relating fluid absorption to the difference in osmolalities in amniotic fluid and fetal plasma. Curved lines are 95% confidence limits of the means. Filled symbols indicate amniotic fluid solutions with albumin.

Oncotic pressure and amniochorionic absorption. To determine whether the rate of fluid absorption was affected by oncotic pressure differences between amniotic fluid and fetal plasma, we infused bovine albumin solutions into the amnion in eight experiments. In seven of the experiments, the albumin was dissolved in NaCl solutions of various osmolalities and one in a mannitol solution. In these experiments, the oncotic gradient that normally strongly favors fetal plasma was reversed, the mean protein concentration being +14.1 ± 3.4 g/l (P < 0.005), higher in the amniotic fluid than in the plasma. In contrast, the mean difference in the protein concentrations of the amniotic fluids and the fetal plasmas in the 13 experiments in which no protein was infused into the amnion was 29.0 ± 2.6 g/l (P < 0.001). The data points of the experiments in which protein was infused into the amnion are shown as filled symbols in Fig. 2.

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Effect of amniotic fluid osmolality on fetal plasma osmolality. Figure 2 shows that the amniotic fluid osmolalities ranged from 53 mosmol/kg hypoosmolal to 98 mosmol/kg hyperosmolal with respect to fetal plasmas of the same experiment. The actual range of amniotic fluid osmolalities was somewhat greater, being from 231 to 403 mosmol/kg because fetal plasma osmolalities shifted somewhat in the direction of amniotic fluid osmolality. However, the range of fetal plasma osmolalities in these experiments was very much smaller than the range of amniotic fluid osmolalities, remaining only between 280 and 305 mosmol/kg, because the fetal plasma osmolality is tightly coupled to the maternal plasma osmolality by equilibration across the placenta.

Fate of infused protein. Because the amounts of protein infused and recovered were known from the products of the concentrations and the volumes, the amounts of protein that were absorbed from the amniotic fluid could be calculated. This absorption rate was 1.8 ± 0.4 g/h (n = 8, P < 0.001). To confirm that the fetal circulation can accommodate the amounts of bovine albumin that were absorbed from the amniotic fluid, we infused a bovine albumin solution of 108 g/l in isotonic Ringer solution directly into the vein of one fetus before any other experiments were performed. After 30 g of protein had been infused in 21 h, fetal plasma protein concentration had risen only from 37 to 42 g/l, and fetal hematocrit had decreased from 35 to 31%. This further agrees with unpublished experiments performed in our laboratory in which homologous protein infused into sheep fetuses was rapidly cleared from the circulation.

Fluid flows associated with protein absorption. We considered it unlikely that the protein that was absorbed was carried by the fluid flows driven by the differences in crystalloid osmolalities, because such flows require the existence of nonzero reflection coefficients for the crystalloids (6, 11). This would be possible only in passages too narrow to permit the passage of proteins. There was the additional consideration that protein concentration differences did not demonstrably affect the water absorption rate, indicating that the protein reflection coefficients in the passages that did allow protein absorption were close to zero. Thus the paths that allowed protein to pass did so without discrimination of molecular size. Because both the protein absorption rates and the protein concentrations were known, the flows of fluid associated with protein absorption rates could be calculated. The mean flow was 33.8 ± 6.1 ml/h (P < 0.001), a value that was not significantly different (P = 0.2) from the intercept of 23.8 ml/h of the linear regression of water absorption as a function of the difference in osmolalities (Fig. 2).

Absence of diffusional transfer of albumin between plasma and amniotic fluid. In three fetuses, ¹²⁵I-labeled human albumin was injected intraveously. Figure 3 shows the concentrations of trichloracetic acid-precipitable label in fetal plasma and amniotic fluid during the next 24 h and beyond. Although there was detectable radioactivity in the amniotic fluid near the ends of these experiments, the amounts were so small that they argued against a diffusional mechanism to account for the absorption of the bovine albumin from the amniotic fluids.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Concentrations of 125I-labeled albumin in fetal plasma and amniotic fluid after a single intravenous dose at time 0 in three fetal lambs. Data normalized by setting plasma concentrations at 19 h equal to 100 and converting other values accordingly.

Perspective. The physiological properties of the additional path of amniotic fluid absorption closely mimic those of the lymphatic system. However, a lymphatic system in the amniochorion has not been described, and the umbilical cord is known not to contain lymphatic trunks (3, 12). Although the force of this argument is diminished by the consideration that conventional methods for demonstrating terminal lymphatics are not likely to work in the amiochorion, the anatomic basis for this path remains obscure.