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Newborn babies are able to control their body temperature by adjusting their metabolic rate, irrespective of ambient temperature(1). This ability is acquired during intrauterine life under highly stable thermal conditions. Although ontogenic changes in endocrine responses to hypotension(2, 3), hypoxemia(4–6), and cord compression(7) have been investigated in the sheep fetus, no conclusive information exists regarding developmental changes in metabolic and endocrine responses during fetal life to cold exposure. Lack of such information is even more surprising considering that an adequate response to the thermal environment is one of the most important prognostic factors for the survival of the premature infant(8, 9).

Thermogenesis is inactive during fetal life(10), and is quickly activated immediately after birth. Previous studies indicated that placental separation and cold exposure are major triggers for this activation of heat production at birth(11), and suggested that circulating placental products may suppress thermogenesis in utero(12, 13). However, there have been no studies to date characterizing the ontogenic changes in fetal intrinsic responsiveness to cold exposure without the presence of placental factors, possibly due to technical difficulties in stably maintaining respiration of premature fetuses separated from the placenta. Recently, it has been reported that ECMO is applicable to control fetal blood gas conditions in chronically instrumented fetal lambs independent of the ewe(14, 15). To date, no physiologic study has been conducted using ECMO as a total respiratory support for the exteriorized fetus without lung respiration.

In the present study, we hypothesized that, in the fetus isolated from the placenta, metabolic and endocrine responses to cold exposure increase with advancing gestational age. To address this hypothesis, we investigated the effects of acute cold exposure on metabolic and endocrine responses of fetal goats at different stages of gestation, using an umbilical A-V ECMO, a technique recently described for long-term extrauterine maintenance of premature animals. The goat was chosen as an experimental animal in this study because it is the only species reported for use in long-term stable extrauterine maintenance of the fetus(16, 17). Details of the extrauterine fetal incubation model have been reported elsewhere(16, 17). This model permits stable maintenance of goat fetuses for up to 1 wk. We conducted experiments inducing transient cold exposure to exteriorized premature goat fetuses, isolated from the placenta and stably maintained in an artificial amniotic fluid container using the extrauterine fetal incubation system with A-V ECMO.

METHODS

Animal preparation. All the experiments were conducted with the approval of our institutional review body. A cesarean section was performed on six pregnant goats with a singleton fetus at 95 to 134 dGA (term 148 d), and their fetuses (weight range 0.5-2.5 kg) were connected to an ECMO circuit. Surgical procedures have been reported in detail elsewhere(16, 18). In brief, hysterotomy was performed under general anesthesia with 2% halothane. Fetal hind legs were extracted until the umbilicus was fully exposed. An umbilical artery and vein were isolated.(Goats have two umbilical arteries and two normal veins.) A polyvinyl catheter(length, 20 cm; outer diameter, 10 Fr) was inserted through an arteriotomy and advanced beyond the bifurcation of the abdominal aorta. Another catheter was inserted into an umbilical vein, with the tip positioned 2 cm beyond the umbilicus. During this procedure, fetal blood gas exchange was maintained through placental circulation via the remaining umbilical artery and vein. An A-V ECMO commenced immediately after the connection of the catheters to the extracorporeal circuit. The remaining pair of umbilical vessels were cannulated and connected to the circuit. The fetus was transferred to an incubator containing artificial amniotic fluid warmed to 39.5°C. The artificial amniotic fluid consisted of an electrolyte solution (Na+, 75 mmol/L; K+, 2.0 mmol/L; Ca2+, 0.8 mmol/L. Ca-, 55 mmol/L) based on the analysis of goat amniotic fluid(16). The total amount of time needed from the hysterotomy until fetal transfer to the incubator was <30 min.

The incubation system. The extracorporeal circuit consisted of an arterial open top reservoir (maximum volume, 25 mL), a roller pump, a nonmicroporous membrane oxygenator made of hollow silicone fibers, a closed inflatable reservoir, and a heat exchanger (Fig. 1). The oxygenator had a functional surface area for gas exchange of 0.5 m2(Senko Medical Instrument Manufacturing Co., Ltd., Tokyo, Japan). Priming volume of the circuit was 200-230 mL. Priming solution consisted of maternal blood anticoagulated with heparin and balanced appropriately for pH, Na+, K+, and Ca2+. Fetal blood from the umbilical artery catheters drained into the arterial reservoir. Blood flow through the circuit was regulated by a flow-control system to maintain a constant blood volume in the arterial reservoir(19). The blood was oxygenated with 100% oxygen and returned to the umbilical veins via a closed reservoir and heat exchanger.

Figure 1
figure 1

Diagram of the extrauterine fetal incubation system:1, heater; 2, incubator and an exteriorized fetus;3, umbilical vein catheter; 4, umbilical artery catheter;5, tube occluder; 6, arterial open top reservoir;7, blood pump; 8, blood surface detector; 9, pump flow controller; 10, membrane oxygenator; 11, venous inflatable reservoir; 12, heat exchanger; and 13, sample ports for preoxygenated and postoxygenated blood. The fetus was placed in a double walled water bath containing artificial amniotic fluid and maintained at 39.0-39.8°C in the baseline condition. The outflow from the artery catheters was drained into an open top reservoir with which blood level detection system was installed. The speed of the rotary pump was controlled to keep the blood level of the reservoir constant. The blood was fully oxygenated at the membrane oxygenator. Pulsation of the blood flow was dampened at the venous reservoir, which also served as an air trap. After being warmed by the heat exchanger, the oxygenated blood was returned to the fetus via the umbilical vein catheter.

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The incubator containing sterile artificial amniotic fluid was placed on a clean bench. All the procedures to the fetus were performed with a strict sterile technique to minimize the risk of infection. A heparin solution (400 U/mL) was continuously infused into the circuit to keep the activated coagulation time between 180 and 250 s. The amount of heparin required was 40-60 U/kg/h. The fetus was left without anesthesia in the incubator filled with artificial amniotic fluid maintained at 39.5°C. No muscle relaxant was given to the fetus. The length of umbilical catheters allowed for spontaneous fetal movement. These settings provided the fetus with a physiologic, thermal, and unrestrained environment. A solution containing 30% glucose, 3% amino acids, and 1.5% soy bean oil was administered to the fetus via the extracorporeal circuit at a rate of 2 mL/kg/h, which amounted to 70 kcal/kg/d. Fetal body weight was estimated at the beginning of incubation from a standard curve using the crown-rump length.

Extracorporeal circuit blood gas exchange adjustments. Initial adjustments of the settings of extracorporeal circuit were completed with repeated measurements of fetal arterial blood gases within the first 24 h of incubation. In this system, fetal PaCO2 is a function of the FIO2, the fetal oxygen consumption, the QEC (mL/min), and the oxygenator performance. Previous studies by other investigators(20) and ourselves(21) revealed the difficulty in maintaining QEC within a physiologic range of umbilical blood flow (approximately 200 mL/kg/min) in long-term incubation of exteriorized fetuses using A-V ECMO. This is because a large QEC makes the fetal circulatory condition unstable. We maintained FIO2 at 1.0 to maximize oxygen delivery with any given QEC which enabled us to stabilize the fetal cardiovascular system by reducing the QEC. The QEC was maintained between 60 and 130 mL/kg/min(21) by altering the impedance of the arterial portion of the circuit using a tube occluder (Fig. 1). Under this condition, the capacity of the extracorporeal circuit to eliminate CO2 was determined by the inspired gas flow/QEC ratio. Fetal PaCO2 was maintained by controlling oxygen flow between 5.3 and 6.7 kPa (40-50 mm Hg). The settings of the extracorporeal circuit were not altered during the cold exposure protocol.

Measurements and calculations. A catheter was inserted into a carotid artery under local anesthesia with 1% lidocaine, allowing aBP to be monitored continuously. QEC was determined with an electromagnetic flowmeter (MFV3200, Nihon Kohden Co., Ltd., Tokyo), attached to the arterial portion of the blood circuit. The HR was counted from the aBP pulse or the umbilical artery wave form. Fetal aBP, QEC, and HR were recorded continuously using a polygraph (no. 362, Nihondenki-Sanei Co., Ltd., Tokyo). Fetal core temperature (°C) was determined with a temperature probe (model BAT 12, Sensortek Inc., Clifton, NJ) chronically implanted into the mediastinal space through an incision made in the neck during the arterial catheter insertion. Blood gas tensions and pH were measured by a 170 pH/blood gas analyzer (Ciba Corning Diagnostics Corp., Medfield, MA, calibrated at 37.0°C). SO2 and Hb concentration were measured with a Hemoxymeter (OSM-2, Radiometer Co., Ltd., Copenhagen) calibrated for goat blood using fully saturated maternal blood. Blood samples from the sample ports at the venous and arterial side of the circuit (Fig. 1)(13) were taken simultaneously at 3- and 6-h intervals for measurement of pH, PCO2, PO2, SO2, and Hb throughout the extrauterine fetal incubation periods. HR, aBP, QEC, and fetal core temperature were recorded during each blood sampling procedure. Preoxygenated (arterial side) and postoxygenated (venous side) CO2, ˙DO2, and fetal˙VO2 were calculated as follows. Equation

Experimental protocol. Cold exposure experiments were started only when all the following criteria were satisfied: 1) >36 h of the extrauterine incubation, 2) stable fetal circulatory variables(aBP >40 mm Hg; HR between 150-230 beats/min; and stable QEC between 60 and 130 mL/kg/min); 3) absence of metabolic acidosis (base excess between -3 and +3 mmol/L); and 4) absence of signs of excessive water retention confirmed with ultrasound scanning.

When the above criteria were satisfied, pre- and postoxygenated blood samples (0.5 mL each) were taken for measurement of blood gases, pH, and SO2. An additional 10 mL of preoxygenated fetal blood were collected for hormone analyses. To avoid a decrease in circulating blood volume, 10 mL of donor blood were infused to the arterial reservoir during sampling.

After baseline measurements, fetal body temperature was reduced by altering fluid temperature of the incubator from 39.5 to 37.5°C. After 2 h of cold exposure, the thermostat setting was returned to 39.5°C. Body temperature was recorded at 15-min intervals during the cold exposure protocol.

The blood sampling was repeated 1, 2, 3, 6, and 12 h after the onset of cold exposure. HR, aBP, and QEC were recorded at each time point of blood sampling.

Cold exposure experiments were repeated if the preparation was stable and all entry criteria (see above) were satisfied. At least 12 h were allowed between any two cold exposure protocols. Two sets of cold exposure experiments were performed on four fetuses and one experiment on the other two. In animals on which repeated cold exposure experiments were performed, averaged data at respective time points were used for statistical analyses, after the lack of changes in baselines and responses was confirmed with the paired t test on the respective time points of each parameter. All fetuses survived for at least 36 h after the last cold exposure experiment.

Hormone analyses. The blood was immediately transferred to an iced tube containing EDTA-disodium and centrifuged at 3000 rpm for 20 min at 4°C. The plasma was stored at -80°C until assayed.

Plasma catecholamine concentrations were measured by a fully automated HPLC-fluorometric system (model HLC-8030 catecholamine analyzer, Tosoh, Tokyo) using the diphenylethylenediamine condensation method(22). The interassay and intraassay CVs were 8 and 5%, respectively. Plasma ACTH levels were measured by an immunoradiometric assay using a ACTH-II IRMA kit (Mitsubishi-Yuka Co. Ltd. Tokyo)(23). The intra- and interassay CVs for the assay were 7 and 6%, respectively. Plasma cortisol concentrations were determined using the SPAC-S cortisol kit (Daiichi Radioisotope Labs., LTD., Tokyo, Japan), which is a reagent set for the solid phase competitive RIA of cortisol that contains tubes coated with cortisol MAb. The intra- and interassay CVs were 5 and 6%, respectively. Plasma T3 and T4 levels were measured by RIAs using the M-T3 Corning and MT-4 Corning RIA kits, respectively (Ciba Corning Diagnostics Corp., Tokyo). The intra- and interassay CVs for T3 and T4 were 3 and 11%, and 5 and 3%, respectively.

Data analyses. Data are presented as SEM. Data were analyzed first by the summary of measures method(24) to focus the statistical comparisons. To analyze ontogenic changes in fetal responses to cold exposure, we conducted two different analyses. In the first analysis(group comparison), the animals were divided into two groups by fetal age(group I, <120 dGA at surgery; group II, >120 dGA at surgery). Physiologic and endocrine changes in each group were analyzed using one-way ANOVA with repeated measures followed by the Student-Neuman-Keuls test. A t test was applied to determine any difference of the baseline variables between the age groups. In the second analysis (regression analysis), in parameters which showed significant changes during cold exposure in either group in the group comparison, maximal changes from baseline were determined at each cold exposure, and their relationship with gestational age was analyzed with regression analyses. Statistical significance was set at the level p < 0.05.

RESULTS

Outcome of Long-Term Extrauterine Fetal Incubation

The incubation period ranged from 91 to 190 h (mean ± SEM, 149± 17 h). Trunk and leg movements were observed in all fetuses within 2 h of the commencement of extrauterine fetal incubation. Breathing and swallowing movements were observed by 6 h. The episodic nature of the presence of these movements suggested the occurrence of behavioral cycles. After 3-4 d of incubation, gradual water retention was noticed as judged by subcutaneous edema and the presence of ascites determined ultrasonographically. All cold exposure protocols were performed before noticing any signs of water retention. Fetuses 1 and 2 died due to repeated catheter occlusions induced by their abrupt movements. Fetuses 3-6 demonstrated a gradual reduction in arterial blood pressure with marked water retention as exemplified by the presence of ascites, pleural effusions, and subcutaneous edema, indicative of circulatory failure (Table 1). Signs of mild infection were found only in the lungs of fetus 6.

Table 1 Summary of extrauterine fetal incubation experiments
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The mean ˙VO2 of all animals during stable conditions was 290± 10 μmol/kg/min (6.4 ± 0.3 mL/kg/min), the value being very close to the ˙VO2 values reported in chronically instrumented lamb fetuses(18).

Fetal Responses to Cold Exposure and Changes in Body Temperature during Cold Exposure

Cold exposure produced a 2°C decrease in body temperature within 45 min (Fig. 2). The body temperature remained stable until the thermostat setting was changed. Fetal body temperature returned to the baseline level in approximately 1 h after cold exposure had been eliminated, and remained constant within the range between 39.0 and 39.8°C throughout the study. No shivering was observed throughout the protocol.

Figure 2
figure 2

Changes in fetal body temperature (BT, mean± SEM, n = 6) during the cold exposure protocol. BT was recorded at 15-min intervals during cold exposure. The BT immediately decreased from baseline after the onset of cooling, and remained constant from 1 h to 2 h. At 2 h, the temperature of incubating fluid was elevated and the BT returned to the baseline level by 3 h.

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Group comparison. Baseline conditions. At 12 h after the commencement of cold exposure protocol, we did not find any change from the baseline in aBP, HR, Hb, SaO2, pHa, PaCO2, PaO2, base excess, ˙DO2, VO2, or QEC in either age group (Table 2, Figs. 3-5). Between two age groups, significant differences at the baseline were observed in Sao2, PaO2, QEC, ˙DO2, and˙VO2, the variables of which were greater in group I. Norepinephrine and cortisol concentrations at the baseline were significantly higher in group II than in group I (Fig. 5), whereas no significant differences were found in epinephrine and ACTH. During cold exposure aBP and HR remained unchanged in both age groups.

Table 2 Fetal conditions at baseline and at the end of cold exposure protocol
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Figure 3
figure 3

Fetal pHa and blood gases. (A) pHa,(B) PaCO2, and (C) PaO2 during cold exposure protocol. *p < 0.05 compared with the baseline value of each age group (one-way repeated measures ANOVA followed by the Student-Neuman-Keuls test). Values are expressed mean ± SEM; □,<120 dGA, n = 3; •, >120 dGA, n = 3.

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Figure 5
figure 5

Fetal endocrine responses, (A) fetal plasma norepinephrine (Nor), (B) epinephrine (Epi),(C) ACTH, and (D) cortisol concentrations, during cold exposure protocol. *p < 0.05 compared with the baseline value of each age group (one-way repeated measures ANOVA followed by the Student-Neuman-Keuls test). Square symbols represent data from group I(<120 dGA); round symbols represent data from group II (>120 dGA). Values are expressed as mean ± SEM when n = 3. Only two sets of data were obtained in Nor and Epi from group I and in ACTH from group II, where data are presented separately without error bars.

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Changes in pH and arterial blood gases during cold exposure. In group I (<120 dGA), no significant changes in pHa or PaCO2 occurred during the experimental protocol. In contrast, a significant decrease in pHa and a significant increase in PaCO2 were measured during cold exposure in group II (>120 dGA) (Fig. 3,A and B). These changes returned to baseline shortly after the body temperature returned to the baseline range. PaO2 remained unchanged from baseline in both group I and II during cold exposure (Fig. 3C).

Changes in oxygen metabolism during cold exposure. Because Hb remained unchanged throughout the experimental protocol, ˙DO2 by the extracorporeal circuit was preferentially determined by the QEC. During cold exposure, ˙DO2 remained unchanged in both group I and II (Fig. 4A). However, a significant increase in ˙VO2 was calculated in group II animals during cold exposure (Fig. 4B). This increase returned to baseline after return of body temperature to baseline levels. Contrary to this,˙VO2 tended to decrease during hypothermia in the younger group, and increased compared with the lowest value of ˙VO2 when the body temperature recovered at 3 h of the protocol.

Figure 4
figure 4

Fetal oxygen metabolism. (A) ˙DO2 and (B) ˙VO2 during cold exposure protocol. *p< 0.05 compared with the baseline (one-way repeated measures ANOVA followed by the Student-Neuman-Keuls test). Values are expressed as mean ± SEM;□, <120 dGA, n = 3; •, >120 dGA, n = 3.

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Endocrine changes during cold exposure. Although plasma norepinephrine, epinephrine, ACTH, and cortisol remained unchanged from baseline by cold exposure in group I animals, a significant increase in the plasma concentrations of norepinephrine, epinephrine, and ACTH occurred during cold exposure in group II animals (Fig. 5,A-C), although changes in plasma cortisol concentrations in group II did not reach to the statistically significant level (p = 0.10) (Fig. 5D). Plasma concentrations of T3 and T4 at baseline in group I were 0.9 ± 0.1 nmol/L and 60.4 ± 15.3 nmol/L (n = 3), respectively. In group II, plasma T3 and T4 levels were 1.2± 0.2 nmol/L and 51.6 ± 8.4 nmol/L (n = 3), respectively. Plasma concentrations of T4 and T3 remained unchanged during cold exposure period.

Regression analysis. Figure 6 plotted the maximal changes in ˙VO2 from the baseline value during cold exposure as a function of gestational age and demonstrated steady increases in responses in terms of ˙VO2. Polynomial regression analysis revealed that there was a significant linear relationship between gestational age andΔ˙VO2. The regression line intersected the x(gestational age) axis at 106 dGA.

Figure 6
figure 6

Changes in the responsiveness of ˙VO2 to ambient cooling during the late gestation in exteriorized goat fetuses. Values are maximal changes from baseline during cold exposure. r2 = 0.90 (p < 0.004).

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Figure 7 illustrates maximal changes in plasma norepinephrine, epinephrine, ACTH, and cortisol concentrations during cold exposure plotted against gestational age. Polynomial regression analysis indicated straight lines as the best fit curves, although the regression analysis in ACTH revealed that the correlation between gestational age andΔACTH failed to reach to the statistically significant level(p = 0.09). The regression lines intersected the x axis at 98 dGA in norepinephrine, 100 dGA in epinephrine, 102 dGA in ACTH, and 98 dGA in cortisol.

Figure 7
figure 7

Changes in the responsiveness of plasma concentrations in (A) norepinephrine (ΔNor), (B) epinephrine (ΔEpi), (C) ΔACTH, and(D) Δcortisol to ambient cooling during the late gestation in exteriorized goat fetuses. Values are maximal changes from baseline during cold exposure. ΔNor, r2 = 0.88 (p = 0.02);ΔEpi, r2 = 0.80 (p = 0.04); ΔACTH,r2 = 0.66 (p = 0.09); ΔCortisol,r2 = 0.70 (p = 0.04).

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DISCUSSION

This study investigated the metabolic, cardiovascular, and endocrine responses to cold exposure in fetal goats of an age too immature to survive unsupported outside the uterus. For this purpose we used an extrauterine fetal incubation system which enabled stable long-term incubations of premature fetuses, allowing investigation of fetal responses to external cooling at different ages. Our findings demonstrated that the development of metabolic and endocrine adaptation to cold exposure occurs in fetal goats of around 100 dGA.

After connection to extrauterine fetal incubation system, fetal cardiovascular responses became stable after 24 to 36 h of incubation as assessed by HR, aBP, pH, and arterial blood gases. The values of variables examined (Table 2, Figs. 3-5) were comparable with those reported for fetuses in utero at baseline, with the exception of heart rate which showed a tendency toward tachycardia. The oxygen delivery was lower than values for fetuses in utero(25) possibly due to the limited capacity of the extrauterine fetal incubation system. The maximum QEC of the system was limited to approximately 350 mL/min because of the catheter size and the blood pump capacity, which caused relatively low ˙DO2 in larger animals. Our previous observations showed that 10 mL/kg/min (450μmol/kg/min) of ˙DO2 was sufficient to maintain constant and stable fetal ˙VO2 with this system(18). In the present study ˙DO2 remained >10 mL/kg/min throughout the experimental period in all the animals. Relatively lower ˙DO2 may have enhanced oxygen extraction inducing decreases in Sao2 and PaO2, and caused a mild fall in ˙VO2. However, metabolic acidosis or a fall in ˙VO2 were not observed during the baseline period in this study. The stability of these metabolic variables suggests that fetal metabolic conditions were maintained after the adaptation of the fetus to the extrauterine fetal incubation environment.

The precooling concentrations of epinephrine, norepinephrine, ACTH, and cortisol were similar to values reported for experiments in chronic preparations(26). Evaluation of the fetal condition before and after the cold exposure revealed no detectable changes in physiologic variables, suggesting that the general condition of the goats was not affected by cold exposure.

Responses of the newborn mammal to environmental cooling include shivering and nonshivering thermogenesis, and endocrine, metabolic, and cardiovascular changes. Most of these responses have been reported to occur during fetal life. Since the first description of a system for cooling a lamb fetus in utero, using tubing coiled around the fetal trunk, by Gunn and Gluckman(27) in 1983, various fetal responses to cold exposure have been investigated. Fetal hypothermia was associated with tachycardia and hypertension(28). During prolonged cooling, fetal hypoxemia and acidosis were reported(27). Cold stimulation of the fetal cutaneous thermoreceptors induced continuous breathing movements that were associated with high voltage electrocortical activity throughout the cooling period(29). However, these studies were performed on relatively mature fetuses. Therefore, the aims of the present study were to assess differences, if any, of metabolic and endocrine responses between exteriorized immature and mature fetuses. Previous reports(4, 10) as well as our own preliminary studies determined that a 2°C reduction in body temperature and cold exposure for 2 h would provide an adequate stimulation for the endocrine system to react in mature fetuses.

During cold exposure, HR and aBP remained unchanged from baseline both in group I and II fetuses; however, ˙VO2 and plasma concentrations of catecholamines, ACTH, and cortisol were significantly increased in group II but not in group I fetuses. These findings suggest that the activation of the endocrine system observed in relatively mature animals was not secondary to cardiovascular changes but possibly due to direct effect of the cooling. The reason why changes in cardiovascular variables were not detected even though there were significant increases in plasma catecholamine concentrations in group II fetuses in unclear. However, it is possible that presence of artificial A-V shunt may dampen changes in the fetal cardiovascular system.

Little is known about the developmental changes in the oxygen metabolism of fetuses and premature neonates. This is largely due to the difficulty of maintaining premature animals in a stable condition after birth, and due to technical problems of measuring oxygen consumption repeatedly in fetuses in utero. Although various techniques have been used to measure fetal oxygen consumption, to our knowledge, the present study is the first to measure oxygen consumption of the cooled premature fetus directly, and to demonstrate developmental changes in ˙VO2 during cold exposure in the fetus. Using an extracorporeal oxygenation system permitted serial measurements of whole body oxygen consumption. The immature human fetal skin has a potential capacity of percutaneous respiration, which may transfer oxygen at a rate of 3-4 mL/min/m2 under atmospheric oxygen pressure(30). However, the oxygen content of artificial amniotic fluid was approximately 2% of the atmosphere. Thus, in the extrauterine fetal incubation system presented in this report, oxygen delivery to the fetus was assumed to be almost completely dependent on the extracorporeal circuit, because the gas exchange at the fetal skin/artificial amniotic fluid interface could be regarded as negligible.

Increases in oxygen consumption during hypothermia have been described in mature neonates(31, 32). The results of the current study support the earlier finding that the system for thermogenesis matures during fetal life(33). We observed differential˙VO2 patterns in immature and mature fetuses. And the linear relationship of maximal changes with gestational age which intersected the x (gestational age) axis at 105 dGA suggests that the development of an adaptive mechanism to cold exposure takes place at around 105 dGA in goat fetuses. The ˙VO2 appeared to remain constant during cold exposure in immature fetuses. In addition, the absence of an increase in˙VO2 may produce an oxygen deficit and a subsequent increase in˙VO2 in compensation for hypoxia after the recovery of body temperature.

In the present study, an increase in CO2 production directly resulted in an increase in PaCO2 and a fall in pH because oxygen gas flow to the oxygenator was not altered after the initial adjustment had been completed. Thus, the CO2 excretion capacity of the extracorporeal circuit was kept constant at any given QEC. A decrease in pH and an increase in PaCO2 observed during cold exposure in group II may, thus, suggest an increased production of CO2 as a consequence of the increased ˙VO2.

It is possible that the large blood pool of the extracorporeal circuit has an influence on the metabolism of the hormones measured. In addition, the blood pool may have a greater effect in smaller animals. However, in this study, the changes in hormone levels were found only in mature fetuses, whereas immature fetuses exhibited constant levels during cold exposure.

The prominent and steady increase in norepinephrine concentrations during cold exposure is in keeping with earlier studies addressing the regulatory actions of norepinephrine in nonshivering thermogenesis(34). A rise in plasma catecholamine concentrations during hypothermia, in particular norepinephrine, has been reported both in neonates and fetuses(28). Increases in norepinephrine concentration are thought to be a direct stimulus to lipolysis in brown adipose tissue(35). The parallel changes in responses in norepinephrine and ˙VO2 to cold exposure with gestational age suggest that the development of norepinephrine responsiveness to cold exposure is one of the main determinants of the increased oxygen consumption resulting from the increased thermogenesis. In addition, gestational age-related increases in the responses to cold exposure also occur in plasma epinephrine, ACTH, and cortisol concentrations (Fig. 7,B-D). It is likely that the adrenal endocrine system also begins to respond to cold exposure at around 100 dGA in the goat fetus.

In sheep, ambient cooling induces changes in the plasma cortisol levels in neonates(36) and fetuses older than 120 dGA(10). The present study indicates that the pituitary-adrenal axis is functioning in the exteriorized goat fetus older than 120 dGA. Furthermore, the pituitary-adrenal axis may be activated by mild hypothermia. The physiologic role of the elevated plasma cortisol concentration in cooled neonatal lambs has to be clarified. However, cortisol intensifies the circulatory responses during hypothermia(37), which suggests that the activation of the ACTH-cortisol axis has a nonspecific but an important permissive effect on systemic responses to cooling.

It is well established that T3 potentiates thermogenic responses in brown adipose tissue by facilitating responses to catecholamines(38). It is possible that developmental changes in plasma T3 concentrations may modify fetal responses to cold exposure. However, because we did not find any significant change in plasma T3 and T4 concentrations in the present study, it is unlikely that T3 plays a significant physiologic role in the ontogenic changes in fetal responsiveness to cold exposure in the goat fetus.

In a preliminary study, we observed clear, visible shivering movements in exteriorized goat fetuses with the extrauterine incubation system during a greater cold exposure. In addition, exhibited shivering in response to cooling in utero in the fetal sheep of 106 dGA has been reported(29). It is not clear why shivering was not observed during cold exposure in this study. However, it may be that a cold exposure causing a fall of 2°C in body temperature is not an adequate stimulus to induce shivering movements in the fetuses of this study. Because we did not perform electromyographic measurements from skeletal muscles, ontogeny of shivering thermogenesis during fetal life is still an unsolved issue. Further studies are required to address this issue.

As judged by the changes in hormonal responses, it appears that the fetus below 100 dGA does not respond to cooling. Furthermore, changes in˙VO2 may suggest that the fetus below 100 dGA reduce thermogenesis in a cold environment. Further studies are required to elucidate metabolic changes in the fetus below 100 dGA. Measuring changes in plasma concentrations of FFA and glycerol, and continuous monitoring of body-water bath temperature differences will provide important information for better understanding metabolic responses in premature fetuses.

In conclusion, using a novel experimental technique of extrauterine fetal incubation with A-V ECMO, our results suggest that maturation of fetal endocrine and metabolic responsiveness to cold exposure develops at around 100 dGA in the goat fetus. This new approach to extrauterine maintenance of premature animals may provide a valuable opportunity to gain insight into the pathophysiology associated with premature birth.