M. G. Ross

Prophylactic amnioinfusion for meconium-stained amniotic fluid

OBJECTIVEnPrevious studies have demonstrated reduced perinatal morbidity in patients receiving amnioinfusion for meconium-stained amniotic fluid compared with control patients receiving no amnioinfusion. Because amnioinfusion for variable fetal heart rate decelerations has become accepted care, we sought to determine the benefit of prophylactic amnioinfusion for meconium compared with standard care, incorporating therapeutic amnioinfusion for variable decelerations.nnnSTUDY DESIGNnNinety-three term patients with moderate to heavy meconium and no variable fetal heart rate decelerations were randomized to immediate prophylactic amnioinfusion (600 ml saline solution bolus followed by 3 ml/min) or to standard care (including therapeutic amnioinfusion for variable decelerations developing later). All babies had DeLee suctioning on delivery of the head. Laryngeal cords were visualized and tracheal suctioning performed when meconium was seen below the cords. Statistical comparisons were performed using Student t test, Fishers exact test, or chi 2 analysis.nnnRESULTSnThere were no significant differences in the incidence of operative delivery, fetal distress, or meconium below the cords or in newborn Apgar scores and umbilical artery gas values between the amnioinfusion (n = 43) and control (n = 50) patients. There were four cases of meconium aspiration, three in the amnioinfusion group, one in the standard care group. The rate of endometritis-chorioamnionitis was higher (p = 0.3) in the amnioinfusion (16%) than in the control group (8%), although time from ruptured membranes to delivery (8.5 hours vs 7.3 hours) and duration of intrauterine monitoring (6.1 hours vs 5.3 hours) were not different.nnnCONCLUSIONSnAlthough amnioinfusion does dilute amniotic meconium, prophylactic amnioinfusion for meconium in the absence of variable decelerations remains controversial. Prophylactic amnioinfusion in term pregnancies did not improve perinatal outcome and increased the risk for chorioamnionitis-endometritis. Together with recent reports, the current data suggest that the benefit of amnioinfusion for meconium-stained amniotic fluid is a result of the alleviation of variable fetal heart rate decelerations rather than meconium dilution.

Bulk flow of amniotic fluid water in response to maternal osmotic challenge

Amniotic fluid volume is regulated by a complex system of fluid exchanges with fetal and maternal fluid compartments. To assess possible hormonal control of amniotic fluid water exchange with the maternal vascular compartment, we studied the effect of intra-amniotic injections of prolactin, vasopressin, or vasotocin on the amniotic to maternal water flux induced by the acute intravenous infusion of mannitol to the pregnant ewe. This mannitol stimulus increased amniotic fluid osmolality secondary to a shift of free water to the maternal vascular/extracellular compartments. Prior intra-amniotic injection of prolactin but not vasopressin or vasotocin blunted the amniotic fluid osmolar response to maternal mannitol infusion. These results suggest that resorption of amniotic fluid water may occur at the chorioamnion and that amniotic fluid prolactin may have a regulatory function in amniotic fluid volume and osmolar homeostasis.

Effect of maternal food restriction on fetal rat lung lipid differentiation program.

Although “fetal programming” has been extensively studied in many organs, there is only limited information on pulmonary effects in the offspring following intrauterine growth restriction (IUGR). We aimed to determine the effects of nutrient restriction on the lung structure and lung lipid differentiation programs in offspring using an animal mode of maternal food restriction (MFR). We utilized a rodent model of 50% MFR from day 10 of gestation to term and then using lung morphology, Western blotting, Real Time RT‐PCR and Oil Red O staining, lung structure and development of the offspring were examined at postnatal days (p) 1, p21, and 9 months (9M). At postnatal day 1, MFR pups weighed significantly less compared to control pups, but at p21 and 9M, they weighed significantly more. However, lung weight, expressed as a percentage of body weight between the two groups was not different at all time‐points examined. The MFR group had significantly decreased alveolar number and significantly increased septal thickness at p1 and 9M, indicating significantly altered lung structure in the MFR offspring. Furthermore, although at p1, compared to the control group, lung lipid accumulation was significantly decreased in the MFR group, at 9M, it was significantly increased. There were significant temporal changes in the parathyroid hormone‐related protein/peroxisome proliferator‐activated receptor gamma signaling pathway and surfactant synthesis. We conclude that MFR alters fetal lung lipid differentiation programming and lung morphometry by affecting specific epithelial–mesenchymal signaling pathways, offering the possibility for specific interventions to overcome these effects. Pediatr Pulmonol. 2009; 44:635–644.

Ovine Fetal Urine Contribution to Amniotic and Allantoic Compartments

The ovine pregnancy has been extensively studied as a model for amniotic fluid metabolism potentially serving as a model for human oligohydramnios. However, it is recognized that the ovine pregnancy contains an allantoic fluid compartment not present in human pregnancies at term. Earlier studies in sheep suggested that the fetal urine contribution to allantoic fluid diminished at term, but without an explanation for maintenance of the significant volume of the allantoic cavity. In the present study we examined the relative fetal urine excretion of 3H-inulin into the allantoic and amniotic cavities in the near-term ovine pregnancy. Amniotic and allantoic volumes, as determined by technetium-labelled dextran and chromium-tagged erythrocyte dilution, were similar although there were significant differences in composition. The allantoic fluid compartment received an equal or greater proportion of fetal urine than did the amniotic fluid during the study period. These results indicate the importance of the allantoic fluid volume and composition in the study of ovine fluid dynamics.

Central Fos expression in fetal and adult sheep after intraperitoneal hypertonic saline

We hypothesized that neural structures, involved in sensing extracellular body fluid composition in adult animals during an osmotic challenge, would show similar patterns of activation in fetal sheep. Eight adult sheep [4 hypertonic saline-treated adults (HYP-A), 4 isotonic saline-treated adults] and six near-term fetal sheep [3 hypertonic saline-treated fetuses (HYP-F), 3 isotonic saline-treated fetuses; 130 days gestation] were prepared with vascular and intraperitoneal catheters. Seventy-five minutes before tissue collection, hypertonic (1.5 M) or isotonic saline was infused via an intraperitoneal catheter to adult (18 ml/kg) or fetal sheep (6 ml/kg). Brains were examined for patterns of neuronal activation (demonstrated by Fos protein expression). HYP-A and HYP-F demonstrated similar acute increases in plasma osmolality (∼10 mosmol/kgH2O) and comparable patterns of Fos expression within the organum vasculosum of the lamina terminalis (HYP-A, 67 ± 2 vs. HYP-F, 63 ± 6; means ± SE) and hypothalamic supraoptic (SON; HYP-A, 107 ± 8 vs. HYP-F, 102 ± 7) and paraventricular nuclei (PVN; HYP-A, 71 ± 18 vs. HYP-F, 124 ± 19). Fewer activated neurons were detected in HYP-A vs. HYP-F within the subfornical organ (HYP-A, 33 ± 8 vs. HYP-F, 91 ± 17) and median preoptic nucleus (HYP-A, 33 ± 5 vs. HYP-F, 70 ± 6). In adults and fetuses, counterstaining for arginine vasopressin revealed that neurons within the SON and PVN respond to osmotic challenge. These findings demonstrate that central osmoregulatory centers in adult and near-term fetal sheep are similarly activated by osmotic challenge.

Ovine fetal swallowing: expression of preterm neurobehavioral rhythms.

Objective: Fetal swallowing contributes importantly to amniotic fluid volume regulation and fetal gastrointestinal maturation. Near-term ovine fetal swallowing occurs in discrete bouts of activity (at approximately 30-min intervals) in association with fetal electrocortical voltage changes. Thus, swallowing rhythms have been hypothesized to be entrained to fetal neurobehavioral states. In the preterm ovine fetus, electrocortical activity does not demonstrate differentiation into high- and low-voltage periods until 120-130 days gestation. We sought to quantify patterns of preterm (114 days, 0.75 gestation) ovine fetal swallowing activity and volume, and, in view of the lack of electrocortical pattern changes, to explore whether swallowing activity was regulated by an independent central pacemaker. Methods: Six singleton ovine pregnancies were chronically prepared with fetal and maternal femoral artery and vein catheters. Biparietal electrocortical electrodes were placed on the fetal skull. Following a minimum 5-day recovery period, fetuses were studied at 114 - 1 days. Patterns of fetal swallowing behavior were quantified by computer analysis of laryngeal-esophageal electromyography (EMG) and thoracic esophageal fluid flow during a 12-h period. Results: Esophageal fluid flow was bidirectional, although antegrade flow predominated, leading to an average fluid acquisition rate of 13 - 3 ml/h (7.3 - 1.8 ml/h per kg) during the 12-h study (302 - 87 ml/day). Propagated esophageal EMG activity, representing coordinated swallows, averaged 56 - 6 swallows/h and correlated well with net esophageal fluid flow. Bouts of swallowing activity ( S 3 swallows/min) averaged 9 - 1 swallows/bout, lasted 1.8 - 1.4 min and accounted for 31 - 4% of the swallowed volume. Despite the absence of fetal electrocortical high-voltage/ low-voltage transitions, there was a 26.1 - 3.9-min interval between periods of swallowing bout activity. Conclusions: Preterm (0.75 gestation) ovine fetal volume swallowed (302 ml/day) and volume swallowed for body weight (175 ml/day per kg) was significantly less than that previously noted at 0.85 gestation (831 ml/day, 274 ml/day per kg, respectively; p < 0.05) although the rates of swallowing activity were similar. The presence of swallowing bout activity at periodic intervals, in the absence of electrocortical differentiation, suggests an intrinsic central pacemaker regulating preterm fetal neurobehavior.

Fos response of fetal sheep anterior circumventricular organs to osmotic challenge in late gestation

We hypothesized that the anterior circumventricular organs (ACVO) and the supraoptic (SON) and hypothalamic paraventricular nuclei (PVN), among other structures that play a role in sensing extracellular body fluid volume and composition in postnatal animals (as demonstrated by Fos protein production by the immediate-early gene c- fos), would show similar activation in fetal sheep during an osmotic challenge. The brains of 10 fetal sheep [6 treated, 4 controls; 129-131 days of gestational age (dGA) = 0.87 gestation] were immunostained for Fos. Seventy-five minutes before tissue collection the dams were given intravenous 20% mannitol (1 ml ⋅ min-1 ⋅ kg-1for 10 min). Subsequently, the ACVO, SON, and PVN were scored for the amount of neuronal Fos immunostaining. The subfornical organ (SFO; 24.5 ± 9.0 vs. 1.7 ± 1.2), the organum vasculosum of the lamina terminalis (OVLT; 26.8 ± 5.6 vs. 7.0 ± 2.0), the SON (39.8 ± 3.0 vs. 0.15 ± 0.1), and the PVN (59.8 ± 7.9 vs. 0.7 ± 0.7) had increases ( P < 0.05) in the average number of Fos-positive cells per field compared with controls, whereas the median preoptic nucleus did not. Double immunostaining for Fos and arginine vasopressin (AVP) or oxytocin (OT) indicated that AVP- but not OT-immunopositive neurons in SON and PVN respond to osmotic challenge. These results demonstrate that the SFO, OVLT, SON, and PVN are activated by osmotic challenge in fetal sheep at 130 dGA.We hypothesized that the anterior circumventricular organs (ACVO) and the supraoptic (SON) and hypothalamic paraventricular nuclei (PVN), among other structures that play a role in sensing extracellular body fluid volume and composition in postnatal animals (as demonstrated by Fos protein production by the immediate-early gene c-fos), would show similar activation in fetal sheep during an osmotic challenge. The brains of 10 fetal sheep [6 treated, 4 controls; 129-131 days of gestational age (dGA) = 0.87 gestation] were immunostained for Fos. Seventy-five minutes before tissue collection the dams were given intravenous 20% mannitol (1 ml . min-1 . kg-1 for 10 min). Subsequently, the ACVO, SON, and PVN were scored for the amount of neuronal Fos immunostaining. The subfornical organ (SFO; 24.5 +/- 9.0 vs. 1.7 +/- 1.2), the organum vasculosum of the lamina terminalis (OVLT; 26.8 +/- 5.6 vs. 7.0 +/- 2.0), the SON (39.8 +/- 3.0 vs. 0.15 +/- 0.1), and the PVN (59.8 +/- 7.9 vs. 0.7 +/- 0.7) had increases (P < 0.05) in the average number of Fos-positive cells per field compared with controls, whereas the median preoptic nucleus did not. Double immunostaining for Fos and arginine vasopressin (AVP) or oxytocin (OT) indicated that AVP- but not OT-immunopositive neurons in SON and PVN respond to osmotic challenge. These results demonstrate that the SFO, OVLT, SON, and PVN are activated by osmotic challenge in fetal sheep at 130 dGA.

Ovine intramembranous pathway permeability: use of solute clearance to determine membrane porosity.

Objective: The contribution of the fetal chorioamniotic membranes (i.e. the intramembranous pathway) to the regulation and maintenance of amniotic fluid (AF) volume and composition has yet to be completely understood. Knowledge of membrane permeability properties is vital to understanding how the intramembranous pathway contributes to the overall maintenance of AF homeostasis. Although there are significant data regarding the regulation of intramembranous water flow, there is little understanding of the regulation of intramembranous solute flow. In the present study, we sought to determine the effect of molecular weight or size of non-polar compounds on intramembranous solute movement in the ovine model. Methods: Five singleton ovine fetuses (117 - 3 days) were chronically prepared with bladder, tracheal, amniotic cavity and femoral arterial and venous catheters and an esophageal occluder. The allantoic membranes were excised. After 5 days recovery, AF volume was calculated by intraamniotic injection of 99 Tc-labelled red blood cells (time m 6 to 0 h). At time 0, AF exchange routes were limited to the intramembranous pathway by inflation of the esophageal occluder and external drainage of fetal urine and lung fluid. Following intra-amniotic injection of creatinine (Cr, 1 g, MW 11 000 Da, 4 Å) and [ 125 I]albumin (RISA, 250 w Ci, MW 69 000 Da, 36 Å), maternal and fetal plasma and AF samples were collected at timed intervals during the subsequent 5 h. AF solute clearance (Cl x) was determined by the changes in AF total solute content. Results: Cr and RISA disappeared from the AF with a corresponding increase in fetal, though not maternal, plasma levels. The mean Cl Cr was significantly greater than Cl RISA (2.0 - 0.3 ml/min vs. 1.0 - 0.2 ml/min; p < 0.04). Conclusion: Solute clearance from the amniotic cavity is inversely proportional to solute molecular weight/size. Although the membrane comprising the ovine intramembranous pathway is size restrictive, membrane pores allow passage of non-polar solutes up to 36 Å. Knowledge of membrane permeability characteristics is essential for the utilization of the intramembranous pathway for fetal therapeutics.

Increased Fetal Colonic Muscle Contractility Following Glucocorticoid and Thyroxine Therapy: Implications for Meconium Passage

The incidence of meconium-stained amniotic fluid increases with advanced gestational age and fetal stress, and meconium passage is likely dependent on fetal colonic muscle function. Antenatal hormone exposure improves fetal pulmonary and cardiovascular function. We hypothesized that in utero exposure to steroid or thyroid hormones effect an increase in fetal distal colonic muscle contractility. In a randomized controlled study 126-day (term 145 days) ovine fetuses were treated with a single ultrasound-guided intramuscular injection of 0.5 mg/kg betamethasone (n = 5), betamethasone plus 60 micrograms/kg thyroxine (n = 5), or saline (n = 7). After 48 h, fetuses (128 days) were delivered, distal colon segments were removed, and peak tension responses to bethanechol (10(-8) to 10(-3) M) characterized in vitro. Peak muscle tensions were significantly greater in fetuses exposed to combined betamethasone and thyroxine therapy (989 +/- 190 g/cm2) than in saline-treated animals (509 +/- 91 g/cm2). There was difference in the maximum tension response between betamethasone alone (559 +/- 75 g/cm2) and the saline animals. The bethanechol ED50 values (2.1 +/- 0.5 x 10(-5) M) were not different among the three groups. Antenatal fetal betamethasone and thyroid hormone treatment increases fetal colonic muscle contractility. We speculate that endogenous or exogenous fetal maturational agents may facilitate the passage of meconium.

Fetal Arginine Vasopressin under Basal and Hypoosmolal Conditions

Blood samples (4 ml) for plasma arginine vasopressin (AVP) measurements were obtained at 3- to 4-hour intervals under basal conditions for 1-2 days from 5 date-bred ewes with chronic maternal and fetal vascular catheters. In addition, 6 chronically catheterized ewes were infused with 2 liters of 0.45% NaCl over 30 min. Fetal and maternal blood samples were obtained before and after the infusion period for measurement of plasma osmolality and AVP concentrations. In the first study, maternal and fetal plasma AVP levels correlated significantly (p less than 0.01, by linear regression analysis) under basal conditions. In the second study, baseline mean (+/- SEM) plasma osmolality was similar for pregnant ewes and fetuses (303 +/- 3.1 and 302 +/- 2.4 mosm/kg, respectively). There was a significant (each, p less than 0.01 by paired t test) decrease from baseline in maternal and fetal osmolality during the 30 min after completion of the hypotonic saline (to 292 +/- 4.7 and 296 +/- 2.4 mosm/kg, respectively). Fetal plasma AVP levels decreased 17 +/- 6% by 30 min following the completion of water loading (1.7 +/- 0.07 to 1.4 +/- 0.16 microU/ml; p less than 0.05). Maternal plasma AVP levels decreased 16 +/- 4% by 30 min after completion of infusion (1.6 +/- 0.14 to 1.38 +/- 0.6 microU/ml; p less than 0.05). These results indicate that maternal and fetal plasma AVP levels correlate under basal conditions and that maternal water loading, which significantly decreases fetal plasma osmolality, significantly suppresses fetal plasma AVP concentrations.